WO2012004346A1 - Genetically engineered stem cells, method of production and uses thereof - Google Patents
Genetically engineered stem cells, method of production and uses thereof Download PDFInfo
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- WO2012004346A1 WO2012004346A1 PCT/EP2011/061515 EP2011061515W WO2012004346A1 WO 2012004346 A1 WO2012004346 A1 WO 2012004346A1 EP 2011061515 W EP2011061515 W EP 2011061515W WO 2012004346 A1 WO2012004346 A1 WO 2012004346A1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0393—Animal model comprising a reporter system for screening tests
Definitions
- the present invention relates to a genetically engineered stem cell in which a specific genetic locus is modified in a way that a vital fluorescent reporter gene is activated upon differentiation of said cell into a specific cell type such as a serotonergic neuron.
- Serotonin (5-HT) is an important neurotransmitter playing crucial role in the regulation of several behavioural and physiological functions such as mood, memory, circadian rhythms and sexual and feeding behaviour. Moreover serotonin is involved in several processes during embryonic development.
- Serotonin is produced by serotonergic neurons that are localized in the raphe nuclei of the brainstem and 5-HT is synthesized through the activity of the rate-limiting enzyme Tryptophan hydroxylase 2, Tph2, which is selectively expressed in serotonergic neurons. From this region, serotonergic neurons provide a widespread axonal network to the whole central nervous system (CNS), from the forebrain to the spinal cord. The importance of the serotonergic system is further evident from the fact that impairment of 5-HT neurotransmission has been causally linked to several neuropsychiatric disorders such as major depression, bipolar disorders, anxiety, schizophrenia, Attention Deficit Hyperactivity Disorder (ADHD) and autism.
- ADHD Attention Deficit Hyperactivity Disorder
- serotonergic neurons provide a widespread innervation throughout the central nervous system, contacting nearly all the brain structures and involving both synaptic and paracrine signaling; ii) serotonergic neurons provide neuromodulatory actions receiving information from other neurotrasmission systems as glutamatergic and GABAergic systems; iii) serotonegic system is involved in several behavioural and physiological functions such as the regulation of appetite, sleep, memory, mood, stress and sexual behaviour; iv) impairment in serotonin neurotransmission is thought to be causally linked to several neuropsychiatric disorders such as major depression, bipolar disorders, anxiety, schizophrenia, obsessive-compulsive disorder, aggression, Attention Deficit Hyperactivity Disorder (ADHD) and autism and v) several drugs used for the treatment of neurospichiatric disorders act on serotonergic system function.
- ADHD Attention Deficit Hyperactivity Disorder
- Embryonic Stem (ES) cells are totipotent cells derived from the inner cell mass of the early mouse embryo. They show two basic features: they can be cultured indefinitely in vitro (self-renewal) and they have the capability to give rise to all cell types (totipotency). For these reasons, ES cells represent an excellent model to study molecular pathways and mechanisms responsible for differentiation toward specific cell types, and lie at the bases of stem cell-based therapies such as regenerative medicine.
- the first approach mimicks the environment that produces neuroectoderm in the embryo by providing appropriate cell-cell interactions and signals through formation of aggregates made up of cells of all the three primary germ layers (embryo id body, EBs);
- the second approach is to deprive the ES cells of both cell-cell interactions and signals by low density culture in serum-free medium, evoking the default mechanism for neural differentiation.
- Fluorescent proteins were used as cell-type specific reporter (US 7,105,344 B2) using a strategy based on the stable transfection of ES cells with construct comprising a DNA sequence coding for a non-cell damaging fluorescent protein and a development- dependent promoter operably linked with said DNA sequence.
- the reporter expression is driven by tissue specific promoters to obtain stable cell in which the reporter gene is expressed in specific cells or tissue.
- a similar approach was used to generate several stem cell lines that are recently commercialized by Millipore (http://www.laboratorytalk.com/news/mll/mll322.html).
- the patent application US 2008/0118913 discloses a method for genetically accessing serotonergic neurons, using BAC -based transgenes.
- the above examples are based on the random integration of the exogenous gene (transgene) in the host genome. Even if the reporter gene is associated to tissue/cell- and time- specific promoter sequence, the expression of the reporter gene can be affected by the insertion site in the host genome.
- the transgene can be integrated in a compact heterochromatic genomic DNA region or in Vietnamese genomic DNA regions, near specific sequences involved in transcriptional regulation. In the former, despite the presence of a promoter sequence that activates the transcription of the transgene, the heterochromatic structure of genome can inhibit the transcription, thus preventing the expression of the transgene or silencing it.
- Pasqualetti et al, 2002 disclose a Hoxa2 knockin allele that expresses eGFP upon conditional cre-mediated recombination.
- Yadav et al, 2009 disclose a lacZ reporter inserted in the Tph2 locus using embryonic stem cell manipulations. Such system does not allow to visualize live processes as fixing of the cell is needed before assessing the presence of the reporter LacZ.
- the application WO 2008/082125 discloses a knock-out vector wherein a gene to be knocked out is replaced from the initiation codon ATG with a reporter gene Open Reading Frame including its initiation codon.
- the present invention uses a vital reporter gene encoding for a fluorescent protein, such as the gene encoding for the enhanced Green Fluorescent Protein (eGFP).
- a vital reporter gene is a gene encoding for a protein that allows the in vivo real time visualization of a cell expressing said protein, without having to prepare the sample. It allows in vitro and in vivo monitoring of timing and spatial regulation of gene expression and following dynamic behaviour of living systems over time.
- cell types differentiating from ES cells have been mainly characterized by immunocytochemistry staining or by in situ hybridization techniques. These approaches require a first step of fixing the sample, thus preventing the possibility to follow cells during dynamic processes.
- Engineering ES cells in particular by targeting a fluorescent reporter gene to specific loci by a knock-in replacement strategy, offers the possibility to generate genetic tools to study several aspects of neuron development such as differentiation, neurite outgrowth and elongation, neuronal function, neuronal electrical properties. Such cells may be used to establish new and more efficient protocol for in vitro differentiation and to test the effects of exposure to molecules such as growth factors or specific drugs.
- the gene targeting strategy indeed prevents transcriptional interference on the reporter gene due to the integration site and it prevents also the concatenamer integration. Moreover, the gene targeting strategy allows the activation and regulation of the reporter gene under the control of the endogenous tissue/cell specific promoter.
- the present invention refers to the gene targeting of a fluorescent protein gene (for instance eGFP) in the genetic locus of the Tph2 gene that is the ideal marker for serotonergic neurons.
- Tph2 is the key and rate limiting enzyme in biosynthesis of serotonin and during development the beginning of the expression of Tph2 gene correlates with the beginning of serotonin synthesis in the terminally differentiated serotonergic neurons. Tph2 expression is not detectable in any other cell type in the central nervous system. For this reason Tph2 is ideally the best specific molecular marker for serotonergic neurons.
- Tph2 is necessary to produce serotonin and is expressed selectively in serotonergic neuron upon their genesis during development.
- the fluorescent reporter gene cDNA is substituted to the Tph2 coding region.
- the fluorescent reporter protein expression responds to the endogenous promoter of Tph2, ensuring accurate and strict regulation of the fluorescent protein expression that mirrors that of the endogenous gene Tph2.
- the present invention taking advantage of molecular genetic techniques, relates to the engineering of mouse or non-human mammalian ES cells so that a fluorescent vital reporter gene, such as that of the enhanced Green Fluorescent Protein (eGFP), is expressed once the cells differentiate into serotonergic neurons.
- eGFP enhanced Green Fluorescent Protein
- Gene targeting of fluorescent protein coding genes in the Tph2 locus consists in inserting the cDNA encoding for the fluorescent protein in single copy in frame with the first methionin codon of the Tph2 gene. In this way, fluorescent protein expression will likely mirror the expression of the endogenous Tph2 gene.
- This feature makes the stem cell a useful sensor for in vitro serotonergic neuron differentiation since the expression of the fluorescent protein is selectively activated in serotonergic neurons from their genesis. Using this approach, thanks to the expression of the fluorescent protein, it is possible to identify serotonergic neurons once they differentiate in culture, allowing a simple and affordable characterization.
- the main advantage of the present invention is the generation and use of a model system that acts as in vitro sensor for differentiation toward serotonergic neuron phenotype.
- the present invention refers to a stem cell (ES cells) or induced pluripotent stem cell (iPS cells) genetically engineered by gene targeting of a vital reporter gene such as the enhanced green fluorescent protein (eGFP), the red fluorescent protein (RFP), the cyan fluorescent protein (CFP) or the yellow fluorescent protein (YFP), in the genetic locus of the Tph2 gene.
- a vital reporter gene such as the enhanced green fluorescent protein (eGFP), the red fluorescent protein (RFP), the cyan fluorescent protein (CFP) or the yellow fluorescent protein (YFP)
- the present invention also refers to the method to generate such a stem cell and their use to study several aspects of neuron development such as differentiation, neurite outgrowth and elongation, neuronal function, neuronal electrical properties. Indeed, thanks to the fluorescent protein activation in serotonergic neurons as soon as they differentiate, the present invention allows to follow not only the progression during serotonergic neuron development in vitro, but also enables to visualize in real time several aspect of neuronal functioning such as changing in neuronal morphology, migration, axonal outgrowth. Moreover the present invention can be useful to examine the effect of several substances and drugs on serotonergic neurons development.
- the present invention facilitates in vitro studies on serotonergic neuron development and functionality both in standard culture conditions and upon exposure to a wide array of specific molecules ⁇ e.g. growth factors or specific serotonergic drugs) providing a simple and reliable method to generate an in vitro model to study the development and the biology of serotonergic neurons.
- the present invention relates also to a method to test the effects of exposure to molecules such as growth factors or specific drugs in high-throughput screening.
- the Tph2 locus is engineered in order to use the regulatory region of the Tph2 gene to drive the expression of the vital fluorescent reporter gene (e.g. eGFP) with a Tph2-like expression pattern.
- the vital fluorescent reporter gene e.g. eGFP
- the possibility to engineer ES cells and the advantages of targeting the fluorescent protein to specific loci by a knock-in replacement strategy allow an accurate strictly genetic regulation of the reporter gene that is expressed within the same cells as the Tph2 endogenous gene.
- the main advantage of this approach is that the exogenous reporter gene (eGFP) cDNA is driven by the endogenous promoter of Tph2 including all the control regions for the transcription of Tph2.
- the present invention provides also a simple and reliable method to follow in vitro serotonergic neuron development and function from differentiating mouse ES cells and induced pluripotent stem cells (iPS cells).
- stem cells can be subdivided and classified on the basis of their potency.
- Totipotent stem cells are produced from the fusion between an egg and a sperm cell. Cells produced by the first few divisions of the fertilized egg cell are also totipotent. These cells can grow into any type of cell.
- Pluripotent stem cells are the descendants of totipotent cells and can differentiate into any cell type except for totipotent stem cells.
- Multipotent stem cells can produce only cells of a closely related family of cells (e.g. blood cells such as red blood cells, white blood cells and platelets).
- Progenitor (sometimes called unipotent) cells can produce only one cell type, but have the property of self-renewal which distinguishes them from non-stem cells.
- Stem cells can also be categorized according to their source, as either adult or embryonic.
- Adult stem cells are undifferentiated cells found among differentiated cells of a specific tissue and are mostly multipotent, capable of producing several but limited numbers of cell types. They comprise also newborn, umbilical cord, placental and amniotic fluid derived stem cells. They are also called somatic stem cells, or tissue stem cells, and are found in differentiated tissues in which, in a controlled manner, they differentiate and/or divide to produce all the specialized cell types of the tissue from which they originate.
- Embryonic stem cells have the potential of becoming all types of specialized cells including germ cells (pluripotency). They have the capability of proliferating indefinitely in culture, under conditions that allow their proliferation without differentiation. Three types of pluripotent embryonic stem cells have been discovered up to now from rodents and humans:
- ES Embryonic Stem cells
- Teratocarcinomas are gonadal tumors containing pluripotent stem cells present within a wide array of tissues derived from the three primary germ layers (endo-, meso-, and ecto-derm). The most used ones are the mouse EC P19 cell lines. These cells don't have a normal karyotype.
- EG - Embryonic Germ cells
- PSCs Primordial Germ Cells of the foetal genital ridge
- ES and EG cells can be injected into blastocysts of recipient mice giving rise to chimeric animals.
- these pluripotent cells can contribute to every cell type, including the germline.
- murine EC cells introduced into embryos colonize most embryonic lineages, but generally do not colonize the germline, with one experimental exception. The inability of EC cells to form functional gametes most likely reflects their abnormal karyotype.
- iPS Induced Pluripotent Stem Cells
- pluripotent stem cells such as embryonic stem cells
- iPS cells are artificially derived from a non-pluripotent cell, typically an adult somatic cell, by forcing expression of specific genes. Production of iPS cells is an important advance in stem cell research, as it may allow researchers to obtain pluripotent stem cells, which are important in research and potentially have therapeutic uses, without the controversial use of embryos.
- Stem cells are a very powerful tool for High Throughput Screening (HTS) technologies since they can be cultured and expanded in vitro for long periods, maintaining the self-renewal property, and they can undergo miniaturization. They allow the use of selectable and inducible markers for the preparation of a pure population ES cells.
- the technology of gene targeting/homologous recombination allows the Knock Out (KO) or Knock In (KI) of specific genes.
- embryonic stem cells can differentiate into any cell type resembling primary cells (since they are non tumoral cells). In this way they offer a natural environment for the targets, they can address complex targets (like multi- subunit ion channels), that are regulated and expressed in a native way. This is a very important improvement since usually in HTS screening the cell-based assays are set up using tumoral cell lines and it is known that this tumoral environment can alter the physiological cell conditions.
- the use of the vital fluorescent reporter gene in the present invention allows to monitor in vivo regulation of gene expression both in time and space and to follow dynamic behaviour of living systems over the time.
- the present invention provide a method which enable the generation of serotonergic neurons in vitro and allow a simple characterization of the living cells that are accessible to functional, molecular and morphological examination techniques during development or during several dynamic cell behaviour.
- the said ES cell provides the advantage of a vital marker such as eGFP to label neurons expressing the Tph2 gene as a consequence of differentiation into serotonergic neurons and therefore to directly visualize serotonergic neuron differentiation.
- a vital marker such as eGFP
- the unique property of the said ES cell that allows following directly the progression of in vitro differentiation makes this tool suitable to easily test different parameters and set new and more efficient protocols for neuronal differentiation.
- the present invention allows the generation of serotonergic cells differentiated in vitro that can be made accessible to electrophysiological analysis, as being fluorescent cells, they are easily discerned. This means an essential simplification of the functional study of these cells. Moreover, the response of serotonergic neurons to specific molecules or drugs used on said cells can be tested using electrophysiological techniques.
- a pure population of serotonin neurons can be isolated.
- serotonergic neurons differentiated in vitro express the fluorescent protein, they can be separated from other cells present in culture by means of a fluorescence activated cell sorter (FACS).
- FACS fluorescence activated cell sorter
- Recovered cells can be used to generate cDNA and protein expression libraries that represent the full transcriptome of genes expressed in serotonergic neurons.
- the response of serotonergic neurons to specific molecules or drugs can be tested in a single cell or in a pure serotonergic neuron population evaluating changing in the expression of specific genes.
- the present invention allows to follow the progression during serotonergic neuron development in vitro, allowing to visualize in real time several aspect of neuronal development and to follow morphological changes during the time such as the emission of cellular processes, neurite outgrowth and neuronal migration.
- the Tph2 eGFP ES cell of the invention is a useful tool to examine the effect of several molecules and drugs on serotonergic neuron development.
- the present invention facilitates in vitro and in vivo studies on serotonergic neuron development and functionality both in standard culture conditions and upon exposure to a wide array of specific molecules (e.g. specific signalling molecules or new drugs) providing a simple and reliable method to visualize the effect on specific process such as neuronal migration or neuronal projection.
- Stem cells find the broadest potential application in cell therapy and regenerative medicine, which refer to innovative medical therapies that will enable to repair, replace, restore and regenerate damaged or diseased cells, tissues and organs.
- Stem cells offer the opportunity of replenishing other cells, acting as the body's own automatic repair system. This ability makes them an ideal treatment for many diseases, and certain types of stem cells have already been transplanted to restore the blood and immune system function of patients with leukemia, lymphoma or other blood disorders. More, research progresses in understanding how the nervous system develops and functions, hold up the promise that with cell therapy will be also possible to repair damaged nervous tissues.
- Tph2 eGFP ES cell line of the invention is a useful tool to understand the unique navigational characteristics of serotonergic neurons that in turn may aid in devising strategies to instill their positive intrinsic attributes into other types of neurons that fare less well following injury.
- Cell therapy thus offers a promising new approach for the preservation or restoration of cellular function, which may have been lost in injuries, degenerative diseases and disorders of the central nervous system.
- the fluorescent protein is selected from the group of: enhanced green fluorescent protein (eGFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP).
- eGFP enhanced green fluorescent protein
- RFP red fluorescent protein
- CFP cyan fluorescent protein
- YFP yellow fluorescent protein
- the stem cell is an adult stem cell.
- the stem cell is a murine or a human adult stem cell.
- the stem cell is a non-human mammalian embryonic stem cell. Still preferably the stem cell is a mouse embryonic stem cell.
- the stem cell is an induced pluripotent stem cell (iPS cell).
- the exogenous vital reporter coding sequence is under the control of a promoter and/or a regulating sequence of the Tph2 gene, encoding for a Tph2 protein of SEQ ID No. 12 (Swiss prot accession number Q0VBT4), or allelic variants thereof.
- It is a further object of the invention a method to generate the stem cell of any one of previous claims comprising the step of inserting the exogenous vital reporter coding sequence into the stem cell genome so that it is under the control of a promoter and/or a regulating sequence of the Tph2 gene, encoding for a Tph2 protein of SEQ ID No. 12, or allelic variants thereof.
- the insertion of the exogenous reporter coding sequence is performed by homologous recombination so that the initiation methionine codon of the Tph2 coding sequence results to be in frame with the reporter gene coding sequence.
- homologous recombination is performed by means of a recombinant vector comprising from the 5' to the 3' end direction:
- stem cell of the invention for medical use. It is a further object of the invention the use of the stem cell of the invention to monitor serotonergic neuron development and/or function.
- the serotonergic development and/or function is monitored by measuring and/or assessing a parameter selected from the group of: cellular morphology, neurite growth, neurite elongation and/or degeneration, neuronal cell death, neurogenesis, neuronal electrical properties, synaptogenesis or expression of molecular serotonergic markers.
- a parameter selected from the group of: cellular morphology, neurite growth, neurite elongation and/or degeneration, neuronal cell death, neurogenesis, neuronal electrical properties, synaptogenesis or expression of molecular serotonergic markers.
- It is a further object of the invention a method to identify a molecule able to induce or to inhibit the differentiation of stem cells into serotonergic neuron comprising the steps of:
- the method is performed by High Throughput Screening.
- FIG. 1 Targeting strategy for knockin replacement of Tph2.
- Diagram showing (a) the wild type Tph2 locus, (b) the targeting vector, (c) the targeted Tph2 eGFP(neo) allele , and (d) the targeted allele upon Flp-mediated recombination, Tph2 eGFP .
- FIG. 2 (a) Oct4 expression in Tph2 eGFP ES cell as assessed by RT PCR analysis. (b,c) Alkaline Phosphatase activity assay on undifferentiated Tph2 eGFP ES cells.
- ESCs WT cDNA from undifferentiated wild type embryonic stem cells
- ESCs Tph2 eGFP cDNA from undifferentiated Tph2 eGFP embryonic stem cells
- diff Tph2 eGFP cDNA from in vitro differentiated Tph2 eGFP embryonic stem cells
- brain cDNA from adult brain).
- FIG. 3 (a,b) Representative eGFP positive cells differentiated in vitro from Tph2 eGFP ES cells are also positive for 5-HT as highlighted by double immunocytochemistry using antibodies specific for serotonin (a) and eGFP (b). c) in vitro differentiated Tph2 eGFP ES cells expressed markers for serotonergic phenotype by means of RT-PCR analysis.
- ESCs Tph2 eGFP cDNA of undifferentiated Tph2 eGFP ES cells, diff Tph2 eGFP cDNA of differentiated Tph2 eGFP ES cells.
- Figure 4 in vitro differentiated Tph2 eGFP ESCsexpressing eGFP reporter gene in living culture allow direct visualization of serotonergic (eGFP positive) neurons under a common epifluorescence microscope.
- eGFP positive serotonergic neurons
- eGFP fluorescence eGFP fluorescence
- c,f merge of eGFP / brighfield.
- ES embryonic stem
- a genomic fragment containing the genomic region adjacent the Tph2 first exon was isolated from a mouse 129/Sv strain- derived ES cell genomic library (Rijli et al., 1993).
- the isolated fragment referred as pTph2-BC (SEQ ID No. 1), corresponds to the BamHI - Clal restriction fragment around the first Tph2 exon including the first Tph2 exon itself the its flanking genomic regions corresponding to 3.3 kb upstream the first ATG codon and about 3.9 kb downstream it.
- SEQ ID No. 1 pTph2-BC
- the targeting vector was prepared replacing 105 nucleotides of exon 1 downstream the first ATG codon encoding 35 amino acids of the enzyme Tryptophan hydroxylase 2 with the cDNA coding for the vital reporter gene eGFP (SEQ ID No. 2).
- Tph2-likQ control of reporter expression using a recombination-based strategy carried out in DY380 bacteria (Yu et al, 2000), the initiation methionine codon of Tph2 gene (Gene accession number: AY090565) was cloned in frame to the eGFP cDNA starting with the second codon.
- a selection marker cassette coding for the gene neo, driven by the phosphoglycerate kinase (PGK) promoter was placed 3 ' to the reporter gene in the opposite transcriptional orientation than the eGFP.
- a strategy based on Flp mediated recombination was used to generate an allele with a removable selection cassette.
- the PGK-neo cassette was flanked by two FRT sites that allow Flp-mediated excision.
- the PGK-neo cassette was subcloned into the Ncol site of the pIRES-eGFP vector (Clontech Cat. No. 6064-1) to generate the peGFP/neo vector.
- the targeting vector requires homology sequences corresponding to the region of the Tph2 genomic locus to promote homologous recombination.
- Homologous recombination in bacteria is a powerful method for fast and efficient construction of vectors for subsequent manipulation of the mouse genome. This approach is based on homologous recombination in E. coli using recombination proteins present in ⁇ phage.
- the DY380 strain contains a defective ⁇ prophage inserted into the bacterial genome carrying genes exo, bet, and gam, whose transcription is regulated by the PL promoter (Copeland et al, 2001).
- exo is a 5'-3' exonuclease that creates single-stranded overhangs on linear DNA. bet protects these overhangs and assists in the subsequent recombination process, gam prevents degradation of linear DNA by inhibiting E.
- the PL promoter is repressed by a temperature-sensitive repressor at 32°C and de-repressed at 42°C.
- a temperature-sensitive repressor at 32°C
- de-repressed at 42°C.
- the first pair of oligonucleotides amplified a 500 bp DNA fragment (SEQ ID No. 3) upstream to the first ATG codon of the Tph2 gene, while the second one amplified a 500 bp fragment (SEQ ID No. 4) 105 nucleotides downstream it.
- the two homology fragments were subcloned 5' to the eGFP reporter gene and 3 ' to the PGK-neo cassette respectively within the peGFP/neo and the p-LA/eGFP/neo/RA vector was generated.
- Tph2 eGFP(neo) targeting construct SEQ ID No. 9
- DY380 cells Yu et al, 2000
- the pTph2-BC see paragraph "Cloning of mouse Tph2 locus" vector containing the genomic region flanking the Tph2 ATG starting codon and the p-LA/eGFP/neo/RA vector.
- SEQ ID No. 9 targeting vector Tph2 eGFP(neo)
- SEQ ID No. 10 first or left homology arm
- SEQ ID No. 11 second or right homology arm
- Electroporation is the preferred method to introduce the targeting vector within ES cells as DNA concentration and cell density can be adjusted to favour vector integration in single- copy. Any commercially available ES cells may be used.
- the cells line were maintained on gelatinized tissue culture dishes in ES cell medium containing 1000 U/ml leukemia inhibitory factor (LIF) according to BayGenomics protocols available online ⁇ http ://baygenomics.ucsf.edu/>.
- LIF leukemia inhibitory factor
- the Tph2 eGFP(neo) targeting vector was purified on a Qiagen column and 2 x 10 7 ES cells/ 0.7 ml of PBS cells trypsinized from 80% confluent plates were added of 30 ⁇ g of linearized targeting vector and electroporated using a Bio-Rad Gene Pulser unit. Electroporated ES cells were transferred in 40 ml of ES cell medium and plated to a confluence of 5 x 10 6 cells per tissue-culture petri dishes 10 cm diameter. After G418 selection, cells that have integrated the targeting vector form colonies about 1 mm in size. Recombinant resistant colonies were picked and replicated in 96-well plates.
- Genomic DNA was extracted from single ES cell clones, digested with specific restriction enzymes and separated according to size by gel-electrophoresis. In particular, genomic DNA was digested with Bgll and a probe upstream the left homology arm (Probe A) was used to screen for proper 5' recombination. Expected size of signal on autoradiography was 12,5 kb for wild-type allele and 7,3 kb for the recombinant one.
- T ph2 eGFP(neo) ES cells were transfected with the pCAGGS-FLPe vector (Gene Bridges GmbH, Cat. No. A201) that expresses Flp recombinase and confers resistance to puromycin.
- the day of the electroporation 12xl0 6 of 80% confluent cells were electroporated in presence of 15 ⁇ g of the pCAGGS-FLPe vector.
- Tph2 eGFP ES cells maintained totipotency
- Tph2 eGFP ES cells showed high levels of Oct4 expression (Fig. 2a) and strong AP staining (Fig. 2b) indicating that they maintained totipotency.
- the strategies can be grouped in two main approaches: the first approach is mimicking the environment that produces neuroectoderm in the embryo by providing appropriate cell-cell interactions and signals through formation of aggregates made up of cells of all the three primary germ layers (embryoid body, EBs); the second approach is to deprive the ES cells of both cell-cell interactions and signals by low density culture in serum-free medium, evoking a default mechanism for neural differentiation.
- the first approach is mimicking the environment that produces neuroectoderm in the embryo by providing appropriate cell-cell interactions and signals through formation of aggregates made up of cells of all the three primary germ layers (embryoid body, EBs);
- the second approach is to deprive the ES cells of both cell-cell interactions and signals by low density culture in serum-free medium, evoking a default mechanism for neural differentiation.
- serotonin and activation of specific molecular markers suggest that serotonergic neurons differentiated from Tph2 eGFP ES cells in vitro.
- Serotonergic neurons differentiated in vitro appear as multipolar neurons and exhibit long projections forming distinct axonal fascicles and synapses. Morphologically they resemble serotonergic neurons present in the raphe nuclei of the mouse brain.
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Abstract
The present invention relates to a genetically engineered stem cell in which a specific genetic locus is modified in a way that a vital fluorescent reporter gene is activated upon differentiation of said cell into a specific cell type such as a serotonergic neuron.
Description
GENETICALLY ENGINEERED STEM CELLS, METHOD OF PRODUCTION
AND USES THEREOF
FIELD OF THE INVENTION
The present invention relates to a genetically engineered stem cell in which a specific genetic locus is modified in a way that a vital fluorescent reporter gene is activated upon differentiation of said cell into a specific cell type such as a serotonergic neuron.
BACKGROUND OF THE INVENTION
Serotonin (5-HT) is an important neurotransmitter playing crucial role in the regulation of several behavioural and physiological functions such as mood, memory, circadian rhythms and sexual and feeding behaviour. Moreover serotonin is involved in several processes during embryonic development.
Serotonin is produced by serotonergic neurons that are localized in the raphe nuclei of the brainstem and 5-HT is synthesized through the activity of the rate-limiting enzyme Tryptophan hydroxylase 2, Tph2, which is selectively expressed in serotonergic neurons. From this region, serotonergic neurons provide a widespread axonal network to the whole central nervous system (CNS), from the forebrain to the spinal cord. The importance of the serotonergic system is further evident from the fact that impairment of 5-HT neurotransmission has been causally linked to several neuropsychiatric disorders such as major depression, bipolar disorders, anxiety, schizophrenia, Attention Deficit Hyperactivity Disorder (ADHD) and autism. The characteristics of serotonergic neurons suggest that this population would be an experimentally good model to investigate neuron development. Indeed i) serotonergic neurons provide a widespread innervation throughout the central nervous system, contacting nearly all the brain structures and involving both synaptic and paracrine signaling; ii) serotonergic neurons provide neuromodulatory actions receiving information from other neurotrasmission systems as glutamatergic and GABAergic systems; iii) serotonegic system is involved in several behavioural and physiological functions such as the regulation of appetite, sleep, memory, mood, stress and sexual behaviour; iv) impairment in serotonin neurotransmission is thought to be causally linked to several neuropsychiatric disorders such as major depression, bipolar disorders, anxiety, schizophrenia, obsessive-compulsive disorder, aggression, Attention Deficit Hyperactivity
Disorder (ADHD) and autism and v) several drugs used for the treatment of neurospichiatric disorders act on serotonergic system function.
Embryonic Stem (ES) cells are totipotent cells derived from the inner cell mass of the early mouse embryo. They show two basic features: they can be cultured indefinitely in vitro (self-renewal) and they have the capability to give rise to all cell types (totipotency). For these reasons, ES cells represent an excellent model to study molecular pathways and mechanisms responsible for differentiation toward specific cell types, and lie at the bases of stem cell-based therapies such as regenerative medicine.
In the last years, several methods have been established to obtain differentiation of ES cells to specific cell types. In particular, several protocols have been described to obtain neuronal differentiation in vitro from ES cells. These strategies can be basically grouped in two main approaches:
-the first approach mimicks the environment that produces neuroectoderm in the embryo by providing appropriate cell-cell interactions and signals through formation of aggregates made up of cells of all the three primary germ layers (embryo id body, EBs);
-the second approach is to deprive the ES cells of both cell-cell interactions and signals by low density culture in serum-free medium, evoking the default mechanism for neural differentiation.
Fluorescent proteins were used as cell-type specific reporter (US 7,105,344 B2) using a strategy based on the stable transfection of ES cells with construct comprising a DNA sequence coding for a non-cell damaging fluorescent protein and a development- dependent promoter operably linked with said DNA sequence. The reporter expression is driven by tissue specific promoters to obtain stable cell in which the reporter gene is expressed in specific cells or tissue. A similar approach was used to generate several stem cell lines that are recently commercialized by Millipore (http://www.laboratorytalk.com/news/mll/mll322.html). The patent application US 2008/0118913 discloses a method for genetically accessing serotonergic neurons, using BAC -based transgenes.
However, all of the above examples are based on the random integration of the exogenous gene (transgene) in the host genome. Even if the reporter gene is associated to tissue/cell- and time- specific promoter sequence, the expression of the reporter gene can be affected by the insertion site in the host genome. The transgene can be integrated in a compact heterochromatic genomic DNA region or in euchromatic genomic DNA regions,
near specific sequences involved in transcriptional regulation. In the former, despite the presence of a promoter sequence that activates the transcription of the transgene, the heterochromatic structure of genome can inhibit the transcription, thus preventing the expression of the transgene or silencing it. In the latter, different scenarios can be possible due to modulation events induced by regulative sequence sets (enhancers) that are external to the transgene, near the transgene integration site. Moreover, the possibility of transgene concatenamer integration in the genomic DNA could affect the transcriptional control of the transgene, leading to reporter gene silencing (Dobie et al, 1997; Garrick et al, 1998). The limiting aspect of this strategy stems from the difficulty of isolate regulative sequence to drive the expression of the reporter gene to recapitulate the expression pattern in the gene of interest.
The solution to all this technical problems is the use of gene targeting approach via homologous recombination in ES cells.
Pasqualetti et al, 2002 disclose a Hoxa2 knockin allele that expresses eGFP upon conditional cre-mediated recombination. Yadav et al, 2009 disclose a lacZ reporter inserted in the Tph2 locus using embryonic stem cell manipulations. Such system does not allow to visualize live processes as fixing of the cell is needed before assessing the presence of the reporter LacZ. The application WO 2008/082125 discloses a knock-out vector wherein a gene to be knocked out is replaced from the initiation codon ATG with a reporter gene Open Reading Frame including its initiation codon.
The present invention uses a vital reporter gene encoding for a fluorescent protein, such as the gene encoding for the enhanced Green Fluorescent Protein (eGFP). In the context of the present invention, a vital reporter gene is a gene encoding for a protein that allows the in vivo real time visualization of a cell expressing said protein, without having to prepare the sample. It allows in vitro and in vivo monitoring of timing and spatial regulation of gene expression and following dynamic behaviour of living systems over time. To date, cell types differentiating from ES cells have been mainly characterized by immunocytochemistry staining or by in situ hybridization techniques. These approaches require a first step of fixing the sample, thus preventing the possibility to follow cells during dynamic processes.
Engineering ES cells, in particular by targeting a fluorescent reporter gene to specific loci by a knock-in replacement strategy, offers the possibility to generate genetic tools to study several aspects of neuron development such as differentiation, neurite
outgrowth and elongation, neuronal function, neuronal electrical properties. Such cells may be used to establish new and more efficient protocol for in vitro differentiation and to test the effects of exposure to molecules such as growth factors or specific drugs.
In the present invention, expression of a fluorescent reporter gene is selectively induced in serotonergic neurons. The gene targeting (knock- in) strategy indeed prevents transcriptional interference on the reporter gene due to the integration site and it prevents also the concatenamer integration. Moreover, the gene targeting strategy allows the activation and regulation of the reporter gene under the control of the endogenous tissue/cell specific promoter. In particular, the present invention refers to the gene targeting of a fluorescent protein gene (for instance eGFP) in the genetic locus of the Tph2 gene that is the ideal marker for serotonergic neurons.
An ideal molecular marker for a specific cell type is the one that defines that particular cell type, i.e a gene that is expressed in that cell type but not expressed in any other cell type. In the brain, the Tph2 gene is the key and rate limiting enzyme in biosynthesis of serotonin and during development the beginning of the expression of Tph2 gene correlates with the beginning of serotonin synthesis in the terminally differentiated serotonergic neurons. Tph2 expression is not detectable in any other cell type in the central nervous system. For this reason Tph2 is ideally the best specific molecular marker for serotonergic neurons.
Tph2 is necessary to produce serotonin and is expressed selectively in serotonergic neuron upon their genesis during development. With this approach, the fluorescent reporter gene cDNA is substituted to the Tph2 coding region. Then, the fluorescent reporter protein expression responds to the endogenous promoter of Tph2, ensuring accurate and strict regulation of the fluorescent protein expression that mirrors that of the endogenous gene Tph2.
SUMMARY OF THE INVENTION
As ES cells have the potentiality to differentiate toward all cell types, the present invention, taking advantage of molecular genetic techniques, relates to the engineering of mouse or non-human mammalian ES cells so that a fluorescent vital reporter gene, such as that of the enhanced Green Fluorescent Protein (eGFP), is expressed once the cells differentiate into serotonergic neurons.
Gene targeting of fluorescent protein coding genes in the Tph2 locus consists in
inserting the cDNA encoding for the fluorescent protein in single copy in frame with the first methionin codon of the Tph2 gene. In this way, fluorescent protein expression will likely mirror the expression of the endogenous Tph2 gene. This feature makes the stem cell a useful sensor for in vitro serotonergic neuron differentiation since the expression of the fluorescent protein is selectively activated in serotonergic neurons from their genesis. Using this approach, thanks to the expression of the fluorescent protein, it is possible to identify serotonergic neurons once they differentiate in culture, allowing a simple and affordable characterization.
The main advantage of the present invention is the generation and use of a model system that acts as in vitro sensor for differentiation toward serotonergic neuron phenotype. The present invention refers to a stem cell (ES cells) or induced pluripotent stem cell (iPS cells) genetically engineered by gene targeting of a vital reporter gene such as the enhanced green fluorescent protein (eGFP), the red fluorescent protein (RFP), the cyan fluorescent protein (CFP) or the yellow fluorescent protein (YFP), in the genetic locus of the Tph2 gene. In this way, the expression of the reporter gene is specific since it is driven by the endogenous promoter of the Tph2 gene which contains all the regulatory sequences (enhancers). The present invention also refers to the method to generate such a stem cell and their use to study several aspects of neuron development such as differentiation, neurite outgrowth and elongation, neuronal function, neuronal electrical properties. Indeed, thanks to the fluorescent protein activation in serotonergic neurons as soon as they differentiate, the present invention allows to follow not only the progression during serotonergic neuron development in vitro, but also enables to visualize in real time several aspect of neuronal functioning such as changing in neuronal morphology, migration, axonal outgrowth. Moreover the present invention can be useful to examine the effect of several substances and drugs on serotonergic neurons development. The present invention facilitates in vitro studies on serotonergic neuron development and functionality both in standard culture conditions and upon exposure to a wide array of specific molecules {e.g. growth factors or specific serotonergic drugs) providing a simple and reliable method to generate an in vitro model to study the development and the biology of serotonergic neurons. The present invention relates also to a method to test the effects of exposure to molecules such as growth factors or specific drugs in high-throughput screening.
In the present invention, the Tph2 locus is engineered in order to use the regulatory
region of the Tph2 gene to drive the expression of the vital fluorescent reporter gene (e.g. eGFP) with a Tph2-like expression pattern. The possibility to engineer ES cells and the advantages of targeting the fluorescent protein to specific loci by a knock-in replacement strategy allow an accurate strictly genetic regulation of the reporter gene that is expressed within the same cells as the Tph2 endogenous gene. The main advantage of this approach is that the exogenous reporter gene (eGFP) cDNA is driven by the endogenous promoter of Tph2 including all the control regions for the transcription of Tph2.
Thus, the present invention provides also a simple and reliable method to follow in vitro serotonergic neuron development and function from differentiating mouse ES cells and induced pluripotent stem cells (iPS cells).
In the present invention, stem cells can be subdivided and classified on the basis of their potency. Totipotent stem cells are produced from the fusion between an egg and a sperm cell. Cells produced by the first few divisions of the fertilized egg cell are also totipotent. These cells can grow into any type of cell. Pluripotent stem cells are the descendants of totipotent cells and can differentiate into any cell type except for totipotent stem cells. Multipotent stem cells can produce only cells of a closely related family of cells (e.g. blood cells such as red blood cells, white blood cells and platelets). Progenitor (sometimes called unipotent) cells can produce only one cell type, but have the property of self-renewal which distinguishes them from non-stem cells.
Stem cells can also be categorized according to their source, as either adult or embryonic. Adult stem cells are undifferentiated cells found among differentiated cells of a specific tissue and are mostly multipotent, capable of producing several but limited numbers of cell types. They comprise also newborn, umbilical cord, placental and amniotic fluid derived stem cells. They are also called somatic stem cells, or tissue stem cells, and are found in differentiated tissues in which, in a controlled manner, they differentiate and/or divide to produce all the specialized cell types of the tissue from which they originate.
Embryonic stem cells have the potential of becoming all types of specialized cells including germ cells (pluripotency). They have the capability of proliferating indefinitely in culture, under conditions that allow their proliferation without differentiation. Three types of pluripotent embryonic stem cells have been discovered up to now from rodents and humans:
- Embryonic Stem cells (ES) which derive from the inner cell mass of the pre-implantation blastocyst stage embryo. They have a normal karyotype. Several types of mouse and
human embryonic stem cells are known and established, like for example the mouse ES TBV2, R1, D3 cells.
- Embryonic Carcinoma cells (EC) which derive from teratocarcinomas. Teratocarcinomas are gonadal tumors containing pluripotent stem cells present within a wide array of tissues derived from the three primary germ layers (endo-, meso-, and ecto-derm). The most used ones are the mouse EC P19 cell lines. These cells don't have a normal karyotype.
- Embryonic Germ cells (EG) which derive from cultured Primordial Germ Cells of the foetal genital ridge (PGCs). They have a normal karyotype but are abnormally genetically imprinted.
ES and EG cells can be injected into blastocysts of recipient mice giving rise to chimeric animals. In chimeric mice these pluripotent cells can contribute to every cell type, including the germline. In contrast, murine EC cells introduced into embryos colonize most embryonic lineages, but generally do not colonize the germline, with one experimental exception. The inability of EC cells to form functional gametes most likely reflects their abnormal karyotype.
Induced Pluripotent Stem Cells (iPS) are similar to natural pluripotent stem cells, such as embryonic stem cells, in many respects, such as expression of certain stem cell genes and proteins, and viable chimera formation. iPS cells are artificially derived from a non-pluripotent cell, typically an adult somatic cell, by forcing expression of specific genes. Production of iPS cells is an important advance in stem cell research, as it may allow researchers to obtain pluripotent stem cells, which are important in research and potentially have therapeutic uses, without the controversial use of embryos.
Stem cells are a very powerful tool for High Throughput Screening (HTS) technologies since they can be cultured and expanded in vitro for long periods, maintaining the self-renewal property, and they can undergo miniaturization. They allow the use of selectable and inducible markers for the preparation of a pure population ES cells. The technology of gene targeting/homologous recombination allows the Knock Out (KO) or Knock In (KI) of specific genes. Furthermore embryonic stem cells can differentiate into any cell type resembling primary cells (since they are non tumoral cells). In this way they offer a natural environment for the targets, they can address complex targets (like multi- subunit ion channels), that are regulated and expressed in a native way. This is a very important improvement since usually in HTS screening the cell-based assays are set up using tumoral cell lines and it is known that this tumoral environment can alter the
physiological cell conditions.
The use of the vital fluorescent reporter gene in the present invention allows to monitor in vivo regulation of gene expression both in time and space and to follow dynamic behaviour of living systems over the time. The present invention provide a method which enable the generation of serotonergic neurons in vitro and allow a simple characterization of the living cells that are accessible to functional, molecular and morphological examination techniques during development or during several dynamic cell behaviour.
In one embodiment, the said ES cell provides the advantage of a vital marker such as eGFP to label neurons expressing the Tph2 gene as a consequence of differentiation into serotonergic neurons and therefore to directly visualize serotonergic neuron differentiation. This gives the opportunity to facilitate studies elucidating development and functionality of serotonergic neurons in vitro both in standard culture conditions and upon exposure to a wide array of specific molecules or new drugs. The unique property of the said ES cell that allows following directly the progression of in vitro differentiation makes this tool suitable to easily test different parameters and set new and more efficient protocols for neuronal differentiation.
The present invention allows the generation of serotonergic cells differentiated in vitro that can be made accessible to electrophysiological analysis, as being fluorescent cells, they are easily discerned. This means an essential simplification of the functional study of these cells. Moreover, the response of serotonergic neurons to specific molecules or drugs used on said cells can be tested using electrophysiological techniques.
With the present invention, a pure population of serotonin neurons can be isolated. As serotonergic neurons differentiated in vitro express the fluorescent protein, they can be separated from other cells present in culture by means of a fluorescence activated cell sorter (FACS). Recovered cells can be used to generate cDNA and protein expression libraries that represent the full transcriptome of genes expressed in serotonergic neurons. Moreover, the response of serotonergic neurons to specific molecules or drugs can be tested in a single cell or in a pure serotonergic neuron population evaluating changing in the expression of specific genes.
Thanks to the fluorescent protein expression in serotonergic neurons from their terminal differentiation, the present invention allows to follow the progression during serotonergic neuron development in vitro, allowing to visualize in real time several aspect
of neuronal development and to follow morphological changes during the time such as the emission of cellular processes, neurite outgrowth and neuronal migration.
Thus, in particular the Tph2eGFP ES cell of the invention is a useful tool to examine the effect of several molecules and drugs on serotonergic neuron development. The present invention facilitates in vitro and in vivo studies on serotonergic neuron development and functionality both in standard culture conditions and upon exposure to a wide array of specific molecules (e.g. specific signalling molecules or new drugs) providing a simple and reliable method to visualize the effect on specific process such as neuronal migration or neuronal projection.
Stem cells find the broadest potential application in cell therapy and regenerative medicine, which refer to innovative medical therapies that will enable to repair, replace, restore and regenerate damaged or diseased cells, tissues and organs. Stem cells offer the opportunity of replenishing other cells, acting as the body's own automatic repair system. This ability makes them an ideal treatment for many diseases, and certain types of stem cells have already been transplanted to restore the blood and immune system function of patients with leukemia, lymphoma or other blood disorders. More, research progresses in understanding how the nervous system develops and functions, hold up the promise that with cell therapy will be also possible to repair damaged nervous tissues. After injury to the Central Nervous System (CNS), reactive astrocytes hypertrophy over time, forming a physical barrier that contributes to a long term dystrophic state in the vast majority of non- regenerating fibers. When transplanted into the injured adult spinal cord, serotonergic neurons can elongate and incorporate remarkably well into the CNS parenchyma where they can bring about a measure of functional recovery. Tph2eGFP ES cell line of the invention is a useful tool to understand the unique navigational characteristics of serotonergic neurons that in turn may aid in devising strategies to instill their positive intrinsic attributes into other types of neurons that fare less well following injury. Cell therapy thus offers a promising new approach for the preservation or restoration of cellular function, which may have been lost in injuries, degenerative diseases and disorders of the central nervous system.
It is therefore an object of the present invention a stem cell able to express an exogenous vital reporter coding sequence upon differentiation into serotonergic neurons wherein the exogenous vital reporter gene coding sequence encodes for a fluorescent
protein.
Preferably the fluorescent protein is selected from the group of: enhanced green fluorescent protein (eGFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP).
In a preferred embodiment the stem cell is an adult stem cell.
In a preferred embodiment the stem cell is a murine or a human adult stem cell.
Preferably, the stem cell is a non-human mammalian embryonic stem cell. Still preferably the stem cell is a mouse embryonic stem cell.
In a still preferred embodiment the stem cell is an induced pluripotent stem cell (iPS cell). In a yet preferred embodiment the exogenous vital reporter coding sequence is under the control of a promoter and/or a regulating sequence of the Tph2 gene, encoding for a Tph2 protein of SEQ ID No. 12 (Swiss prot accession number Q0VBT4), or allelic variants thereof.
SEQ ID No. 12: Tph2 protein
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It is a further object of the invention a method to generate the stem cell of any one of previous claims comprising the step of inserting the exogenous vital reporter coding sequence into the stem cell genome so that it is under the control of a promoter and/or a regulating sequence of the Tph2 gene, encoding for a Tph2 protein of SEQ ID No. 12, or allelic variants thereof.
Preferably the insertion of the exogenous reporter coding sequence is performed by homologous recombination so that the initiation methionine codon of the Tph2 coding sequence results to be in frame with the reporter gene coding sequence.
Still preferably the homologous recombination is performed by means of a recombinant vector comprising from the 5' to the 3' end direction:
- a first homology nucleotide sequence arm comprising SEQ ID No. 10;
- a sequence wherein the initiation methionine codon of the Tph2 coding sequence is in frame with the reporter coding sequence;
- a selectable cassette sequence that is removable through the action of site specific recombinase, and
- a second homology nucleotide sequence arm comprising SEQ ID No. 11.
It is a further object of the invention the stem cell of the invention for medical use. It is a further object of the invention the use of the stem cell of the invention to monitor serotonergic neuron development and/or function.
Preferably the serotonergic development and/or function is monitored by measuring and/or assessing a parameter selected from the group of: cellular morphology, neurite growth, neurite elongation and/or degeneration, neuronal cell death, neurogenesis, neuronal electrical properties, synaptogenesis or expression of molecular serotonergic markers.
It is a further object of the invention a method to identify a molecule able to induce or to inhibit the differentiation of stem cells into serotonergic neuron comprising the steps of:
a) providing the stem cell as described above;
b) exposing said cell to a compound library comprising putative inducing or inhibiting differentiation agents;
c) detecting and/or measuring the fluorescence.
Preferably the method is performed by High Throughput Screening.
The present invention will be now described by means of non-limiting examples, referring to the following figures.
Figure 1 : Targeting strategy for knockin replacement of Tph2. Diagram showing (a) the wild type Tph2 locus, (b) the targeting vector, (c) the targeted Tph2eGFP(neo) allele , and (d) the targeted allele upon Flp-mediated recombination, Tph2eGFP . (e) Southern blot analysis of recombinant Tph2eGFP(neo) ES cells. DNA was digested with Bgll enzyme and probe A, external to the left homology arm, is used to screen for the 5 ' recombination. The expected size of the wild type and recombinant band is 12.5 kb and 7.3 kb respectively. Genomic DNA digested with Stul was analyzed with probe B external to the right homology arm to screen for the 3' correct recombination. The expected size of the wild type and recombinant band is 8 kb and 5.6 kb respectively.
Figure 2: (a) Oct4 expression in Tph2eGFP ES cell as assessed by RT PCR analysis. (b,c) Alkaline Phosphatase activity assay on undifferentiated Tph2eGFP ES cells. (ESCs WT: cDNA from undifferentiated wild type embryonic stem cells, ESCs Tph2eGFP: cDNA from undifferentiated Tph2eGFP embryonic stem cells, diff Tph2eGFP: cDNA from in vitro
differentiated Tph2eGFP embryonic stem cells, brain: cDNA from adult brain).
Figure 3: (a,b) Representative eGFP positive cells differentiated in vitro from Tph2eGFP ES cells are also positive for 5-HT as highlighted by double immunocytochemistry using antibodies specific for serotonin (a) and eGFP (b). c) in vitro differentiated Tph2eGFP ES cells expressed markers for serotonergic phenotype by means of RT-PCR analysis. (ESCs Tph2eGFP: cDNA of undifferentiated Tph2eGFP ES cells, diff Tph2eGFP cDNA of differentiated Tph2eGFP ES cells.
Figure 4: in vitro differentiated Tph2eGFP ESCsexpressing eGFP reporter gene in living culture allow direct visualization of serotonergic (eGFP positive) neurons under a common epifluorescence microscope. (a,d) brightfield, (d,e) eGFP fluorescence, (c,f) merge of eGFP / brighfield.
DETAILED DESCRIPTION OF THE INVENTION EXAMPLES
Generation of the Tph2 targeting vector
Cloning of mouse Tph2 locus
Engineering of embryonic stem (ES) cells begins by designing a suitable targeting vector to introduce exogenous DNA via homologous recombination within a specific genomic region. In order to generate the targeting vector, a genomic fragment containing the genomic region adjacent the Tph2 first exon was isolated from a mouse 129/Sv strain- derived ES cell genomic library (Rijli et al., 1993). The isolated fragment, referred as pTph2-BC (SEQ ID No. 1), corresponds to the BamHI - Clal restriction fragment around the first Tph2 exon including the first Tph2 exon itself the its flanking genomic regions corresponding to 3.3 kb upstream the first ATG codon and about 3.9 kb downstream it. SEQ ID No. 1 : pTph2-BC
ggatcctcgagtataaattgcttctcagcaactttattgataactcggctttcttatgtct gcaaagtctttgatcattcttttatatcccgttaagcttctagtttttgttgctgatactt gtgaacttatattatttttgtatgcctcaaaaagaaaaggaggccttttatgtttttccat attttttaagtgaacaaaagtggttacagtattgaatagactcattaaatgccttaaatta actttaacattatttaaagcattttatatagtattcttatataatttattttgttcatatt tttctcctttcccaattcctcgcatgtccttaccacttcctcatctaccctattttatctt ctcttcctctcactcttcctcaaaaacataccccaaactgaaaacaaaaagagagcaaaac aaaaaataaagatcagaacaaatgatataaaaacattaggacaaaaaataccaaaactaaa taaaaagcacacagacacacccacacagacacacacccacacagacacacacacacacaca cacacacacacacacacacacacacacacacacagggagttcattttatgttggccaactg cttctgggcatggagcctgccctaaagtatgattgatgtacccaatgatgtaaacagattt ttcctttcttagaaagtatcacttgcaaatagcttcttggctagggataggactttgtgtc
catttcctctcactggtctgaaatgtttgtctggtttggatttgtgcatgctttcgcagtc tctgtgagtttatatgtgcagtggacttgcagtgcttggataaaagtccttggagacatcc cccacctgtagctcagacactcttcctgctttctcagcattgataccttgaaggaaaatgt ttgataaagccatttcatttagtgatgactgttccagagtccctcattctctgctcattgt ccattgccggtctctgtcttaattagcatctactccaagaagcttccctggtgagagctga gtggtccactgatgtacagttacagcattctttcaccagttcccatgctcatcagggacaa tagcctctggacttttaccacttcacatttctggtttctttctttgtattgttaatctacc gtcttaaaccaggctgttgtattctgggcaggatattttaagtccaactttctatcttcca tcttgaagtcctcttccaaggtactagtgttctaaaatgctactatgtttacatgactgca ctgtagaaagcctttccaatcaactttcagttacatgaagaggtgataaagtgccaggctg tgcagcatcatggaaatcttgtcatacgctagatggtgcttctcttttaaatttgtcatcc tttccctacatctagattacaccattttcgaacaataatggtttcctacacattttgttat gttgttccctatattttagaccatccccttgctgttttccattacctatgacactgccaaa atcacttttaaaaaattcagttcaaataactccctcaagagtcttaggactccatagcata tgtagtactgaccactctgtgtcttgtgctttttgtgtcttctgcagagaggttgacactc tttgttctacttggttattgcacagcttgattctctcagatggactgtgatctctgtgggt cagtgctgtgttggacacttctttgcatcctcaggattcagttcccaatgcttaatataaa ctcagagctcaacaaaaagtttttgagttatacatctgttcttatgtatagctctatagtc tgtgatccgttgagtccacttttcttcacacccttctccttatttggagtacttggggaga ttgtacagtgtaatggtagagaaagccaaaattgcctgaattaaaatcctactttgaacat tctttatttgtgtgagcacgtgaacctttgttaatttacctacagcttttgtgccttagta tacatacacacatatatacacacacatgcatacacacacacacacacacacacacacacac acacacacacacacacaatcagtaactctttgtgagtgttgttatgatgactgttctgaca tacaaacatgcatagaaagtgtccagaattagcattgtcaaccatcatttttcataattac ttcggttcatttattaacacttgcttttcctattagctgctcagtgctggttttcagagga caagtaagctggatgattatttccagagttggatcaaacaaaaatcctaaaccctcaagtc tccagaaacaattaaaatccaaccaatgttaacatatacagtcttgctttgaagaaccttc attctgctttctacataatcaaactgtataggtcccagattttggttagttcttcagttaa acatgtgctcttgcttaagaattttccattcttcaaaggattgtcaagctttcctgtggct ttctaaagttggaaaagtacaaatataatcttgtctatgcctgtcaaattgctgggtctga tcaggtcatagatggagagcaataaaattgtatcagaagagtatcaaaggaatgatgggcc tatgggcatttcatttccatatatttcttacttaatgaagaacattctagaaggttagcat ctgagctaagttcaagcctcgaatttcaaaagcctgaatatccttctacatggaaacacca tgggttttggaagcagacttcttatgaaagccgtcacacacacacacacacacacacacac acacacacacacacacacacaggtaaaatttgcataatgtaacgtcatgccagcagaaaag cctttcttaagttatttttttacttttataaaatacaaagcacttaaattcatagttactt aaagacggagggtaaccttccaatagcagacttgagattcaaaattagacgacggctgttt tgttgcaagaaaaaaaaataataaccctgatgtattgttcccctccatctcttcccaaaga gctactcgacctacgaaacaaacaaatctcatcaggagcacagataaccccaagcttcaga cgtgtaatctgactgtggccatcagcaaccagaaatgagttttttctaatcagctttccat cagtcctcagtcactcatataaaggaacacggggaggggaggaagcgcactgctcttcagc accagggttctggacagcgccccgagcaggcagctgccactgcagttcctccttcatctct gccaaggccgcccctctggtcccccctgctgctgagaaagaaaattacatcgggagccatg cagcccgcaatgatgatgttttccagtaaatactgggccaggagagggttgtccttggatt ctgctgtgccagaagatcatcagctacttggcagcttaacagtgagtatcgagtatctggc agtctccggtgttctctagggcatcaatggtcttaacctcaccatatggacacctgctgta gagtaagcatgaagttcaactcccttttcctcagtcgtcttgtcttactgcctcctttctg tctgcctcagctctccacaaagtacaagcagctgggcgagttgctgggagataaactctcc tcaaatagaaagtgaagttccctatcgtgaagaatgaaagattatgtgaaccaatgtgcta aattcactcctccttcaaagtatagaacttgaaaggctgataaggaagatttgggagaaag
aattaaatacatctctctggtttctacaaggcaatattgtctcctatgccctacagagcta atatgtgtattcttgagattaagtgcaggttttggaataaaagaggaaccagggcccttca aaggtttccctgcatgccacattactctccctttaaagcttaattgatggaagagttaagg aagcattctgtttgcttcaatatttacaagatgtgtgtgtgtgtgtgtgtgtgtgtgtgtg tgtgtgtttaaaatactaacattaaagaatggtatttgaagagttacagttcttgcaaagg tttaggtcttaatttgaccaacagttatgagctttaataaacacagtggtttgatttgcta acatgcaataaaatacacatccaaggttctgtcattaaaattttaagggatgcttcaccgt tgtccttcctgtttatcaaccaattgcaaattcagctcgtcagagaaaaattgggttaatg ccatagctccaagtatgtccattctatttattacccacaaacgctgaaaaacagtcacagc aactttcttagcactatttaagtcgatttgcgaaaatgaggccaaaatgtaaaatagcttt tctaatactcaagtatcatgaatatttgtatgaggtgttacattgaggaattccgaagctg taataaacggcaagaagaaaaccacgaagcccttcagttatggttaatgtatgacactttc ccataaaaatgcaccttagacatatgagacacaggcattacctggagaggggggagcagat gttgggaataggaagacatttgagcttctaatggctaaatatactgtcaagtaccagtgcc agcataaactccttctctccctcaactcataaataataaaatatctattcctattatatat aaactcggtgagggaggttatcttaatgcatggaaataaagccagtagtgaaataggagga agacaatggtaaatcacttaccactgggttccatgtaaatgtgcaggtcttagttccacga ggatgtagtagactggtatgactatggtacctcaccattagccaggaagacagatctctgt ttgcccaactctgcaccataatcttacattgtctgtcaaatgggacattcctaggtttgtt ttttcaagttctgcagtgtgctctgatttttgctagactctgttattcctatccttagctg gaagaacttagtcattactcagattgtcgtgtgtgcccctaatggttagctagctggtttt ctttctctctcatgcctttccaactgcctgtcctatatgtggctgaaacgtagtagtcact ttccagactcaactgtccacggagggattcactgggcacgctgctctgtcactgactgcct cagtgagcaggaagattcagacttgggtccaaaggcaactccactttgagttggtatgatg ggtggggcttgcagcttttaagaggaatctctggataaattgaatcattgaaacctctggg gtagaactgactcttgtgcttctactttcttgaagcaaaataaggctatcaaaagcgagga caagaaaagcggcaaagagcccggcaaaggcgacaccacagagagcagcaagacagcggta gtgttctccttgaagaatgaagttggtgggctggtgaaagcacttagactattccaggtaa acacagagcactcgttcttctgtatcctcaaggtggctatgtgcctggtgatagacttgtt ccctgtcattaaggcatttctgggtaacatggcaaaagagtcaaaggaaccaaatgtcaca aagctttaaaaagggagtttactctgcccaacagtgaaataagcaggtttatgtgagctgt gatttattaattcagactcagccaatagcaactctgttacttgttgccagatgaagtctgt aattcaaaaatgtgagctttaaagtagaaaatgcaacacacacacatacatacaatacaca catacatatagacaaaacacacatacacagaacacacacatacacatagacaaaacacata cacagaacacacacatacacacacaatgcatatacacaaaacacacatacctatatacata cacacacaacacacaacacacatatacatacatatacatatacacatatacagaatacaca catatacacaatatacacatacacatagacaaaacacacatacacagaacacacacatacc caacagacatatacacacacaacgcacatacaaaacacacatacctacacacacatataca cacaacacactcacaacacacatatacatgcatacacacatacacacgtatatatacaaca cacatatacacatagacaaagcacacatacacaaaacacacatacctacagacacatacac acacaacacacatatacttatacacacacagacacacatatgcacacacacacacagtgct tcaaaatgccaagagtctttatcctgctttgatttactaaactttaacagtacacatgtca tgagagcatatttgttgcataaaatgagatatatgagcacaggcacttattctcgtaagcc tcatttcatgaacgtaatcgaaggagtgtcttatagaaagatatgccacggtccctggcac gagtccttgttttcagtcctaaatgaagagcttgggtgaggaggaacaccttgtccacctg gtgtcttctccacctgaagcaaaaaaccctcttacaaactacatgtggttgggagcgatgg ctggtcaccagggctgtaagataaacagttcaactctttctggtgccttaggctgctgtgt agcagacacatagttaggggatttggacctgtgttctgctgggttggctaactgctctgtg cacctcggtttgatttcctcatttgtaaaatggcaatgataccaattcctacctaactggc ttgtttggatgaggcgattttgtaaaatgcagacaggaggcattatttgtgatgcctctag
atgaccatagattgggaattaacctgactcggaaaatgtttccttgttcccctttggcctt gaagaaaggaccaggcaccataaaccgtgttaatggaaatctagagtagtattttacacat aattatgccatgtagggctgtgtccacatggtgcccgaaggcaagccaattcaatttgaca gctgggtgcagtgggaaacatccctggggaaggggcttgcactttgaatttgcactctgcc atcagctattcaccaggtctcagctaaaccacttaacccattgagatctcagtgcccagtc taaaaatacttactctgattcaaccaaaccttaaaagcccaaaatggcacaatgtacatgg agtctcttgggccttgaacttaatatcgat
Generation of Tph2e(jtp<neo> targeting vector
The targeting vector was prepared replacing 105 nucleotides of exon 1 downstream the first ATG codon encoding 35 amino acids of the enzyme Tryptophan hydroxylase 2 with the cDNA coding for the vital reporter gene eGFP (SEQ ID No. 2).
SEQ ID No. 2: eGFP cDNA
atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacg gcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacgg caagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctc gtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagc acgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaa ggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaac cgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctgg agtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaa ggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactac cagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagca cccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagtt cgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaagcggccctaga gctcgctgatcagcctcgactgtgcctctagttgccagccatctgttgtttgcccctcccc cgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaa attgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggaca gcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggc ttctgaggcggaaagaaccagctggggctcga
To allow accurate Tph2-likQ control of reporter expression, using a recombination-based strategy carried out in DY380 bacteria (Yu et al, 2000), the initiation methionine codon of Tph2 gene (Gene accession number: AY090565) was cloned in frame to the eGFP cDNA starting with the second codon. A selection marker cassette coding for the gene neo, driven by the phosphoglycerate kinase (PGK) promoter (Rodriguez et al, 2000), was placed 3 ' to the reporter gene in the opposite transcriptional orientation than the eGFP. In order to prevent possible interference of the selection marker on the Tph2 transcriptional regulation, a strategy based on Flp mediated recombination was used to generate an allele with a removable selection cassette. The PGK-neo cassette was flanked by two FRT sites that
allow Flp-mediated excision. The PGK-neo cassette was subcloned into the Ncol site of the pIRES-eGFP vector (Clontech Cat. No. 6064-1) to generate the peGFP/neo vector. The targeting vector requires homology sequences corresponding to the region of the Tph2 genomic locus to promote homologous recombination. Because of the absence of a restriction site close to the first ATG codon of the Tph2 gene suitable to clone the eGFP cDNA in frame with the Tph2 coding region, the traditional restriction endonuclease/T4 DNA ligase strategy for DNA engineering could not be used. To insert the eGFP cDNA and PGK-neo cassette within the Tph2 genomic fragment corresponding to the homology arm sequences an alternative approach based on homologous recombination in E. coli (Yu et al, 2000) was used.
Homologous recombination in E. coli
Homologous recombination in bacteria is a powerful method for fast and efficient construction of vectors for subsequent manipulation of the mouse genome. This approach is based on homologous recombination in E. coli using recombination proteins present in λ phage. The DY380 strain contains a defective λ prophage inserted into the bacterial genome carrying genes exo, bet, and gam, whose transcription is regulated by the PL promoter (Copeland et al, 2001). exo is a 5'-3' exonuclease that creates single-stranded overhangs on linear DNA. bet protects these overhangs and assists in the subsequent recombination process, gam prevents degradation of linear DNA by inhibiting E. coli RecBCD protein. The PL promoter is repressed by a temperature-sensitive repressor at 32°C and de-repressed at 42°C. When bacteria containing this prophage are kept at 32°C no recombination proteins are produced. However, after a brief heat-shock at 42°C a sufficient amount of recombination proteins is produced.
Homologous recombination in E. coli DY380, as well as homologous recombination in ES cells, required the preparation of a targeting construct. Polymerase chain reaction (PCR) was performed to generate two homology 5' and 3' fragments (SEQ ID No. 3 and SEQ ID No. 4, respectively), by using two couples of specific oligonucleotides. SEQ ID No. 3 : 5 ' homology fragment
cataatgtaacgtcatgccagcagaaaagcctttcttaagttatttttttacttttataaa atacaaagcacttaaattcatagttacttaaagacggagggtaaccttccaatagcagact tgagattcaaaattagacgacggctgttttgttgcaagaaaaaaaaataataaccctgatg tattgttcccctccatctcttcccaaagagctactcgacctacgaaacaaacaaatctcat caggagcacagataaccccaagcttcagacgtgtaatctgactgtggccatcagcaaccag
aaatgagttttttctaatcagctttccatcagtcctcagtcactcatataaaggaacacgg ggaggggaggaagcgcactgctcttcagcaccagggttctggacagcgccccgagcaggca gctgccactgcagttcctccttcatctctgccaaggccgcccctctggtcccccctgctgc tgagaaagaaaattacatcgggag
SEQ ID No. 4: 3' homology fragment
agtatcgagtatctggcagtctccggtgttctctagggcatcaatggtcttaacctcacca tatggacacctgctgtagagtaagcatgaagttcaactcccttttcctcagtcgtcttgtc ttactgcctcctttctgtctgcctcagctctccacaaagtacaagcagctgggcgagttgc tgggagataaactctcctcaaatagaaagtgaagttccctatcgtgaagaatgaaagatta tgtgaaccaatgtgctaaattcactcctccttcaaagtatagaacttgaaaggctgataag gaagatttgggagaaagaattaaatacatctctctggtttctacaaggcaatattgtctcc tatgccctacagagctaatatgtgtattcttgagattaagtgcaggttttggaataaaaga ggaaccagggcccttcaaaggtttccctgcatgccacattactctccctttaaagcttaat tgatggaagagttaaggaagca
The first pair of oligonucleotides amplified a 500 bp DNA fragment (SEQ ID No. 3) upstream to the first ATG codon of the Tph2 gene, while the second one amplified a 500 bp fragment (SEQ ID No. 4) 105 nucleotides downstream it.
Primers used for were the following:
5'- ATTATTGGATCCGAATTCATAATGTAACGTCATGCCAGCAGA-3' (SEQ ID No. 5) 5'- ATTATTCCATGGCTCCCGATGTAATTTTCTTT-3' (SEQ ID No. 6)
5'- ATTATTCTCGAGTATCGAGTATCTGGCAGTCTCCG-3' (SEQ ID No. 7)
5 '- ATTATTGTCGACGAATTCTGCTTCCTTAACTCTTCCATCAATTA-3 ' (SEQ ID No. 8)
The two homology fragments were subcloned 5' to the eGFP reporter gene and 3 ' to the PGK-neo cassette respectively within the peGFP/neo and the p-LA/eGFP/neo/RA vector was generated.
In order to obtain a Tph2eGFP(neo) targeting construct (SEQ ID No. 9), DY380 cells (Yu et al, 2000) were co-electroporated with the pTph2-BC (see paragraph "Cloning of mouse Tph2 locus") vector containing the genomic region flanking the Tph2 ATG starting codon and the p-LA/eGFP/neo/RA vector. SEQ ID No. 9: targeting vector Tph2eGFP(neo)
Legend :
BLACK: left homology arm
underline: eGFP
Bold: pgk neo
cursive : right homology arm
ggatcctcgagtataaattgcttctcagcaactttattgataactcggctttcttatgtct gcaaagtctttgatcattcttttatatcccgttaagcttctagtttttgttgctgatactt gtgaacttatattatttttgtatgcctcaaaaagaaaaggaggccttttatgtttttccat attttttaagtgaacaaaagtggttacagtattgaatagactcattaaatgccttaaatta actttaacattatttaaagcattttatatagtattcttatataatttattttgttcatatt ctcttcctctcactcttcctcaaaaacataccccaaactgaaaacaaaaagagagcaaaac aaaaaataaagatcagaacaaatgatataaaaacattaggacaaaaaataccaaaactaaa taaaaagcacacagacacacccacacagacacacacccacacagacacacacacacacaca cacacacacacacacacacacacacacacacacagggagttcattttatgttggccaactg cttctgggcatggagcctgccctaaagtatgattgatgtacccaatgatgtaaacagattt ttcctttcttagaaagtatcacttgcaaatagcttcttggctagggataggactttgtgtc catttcctctcactggtctgaaatgtttgtctggtttggatttgtgcatgctttcgcagtc tctgtgagtttatatgtgcagtggacttgcagtgcttggataaaagtccttggagacatcc cccacctgtagctcagacactcttcctgctttctcagcattgataccttgaaggaaaatgt ttgataaagccatttcatttagtgatgactgttccagagtccctcattctctgctcattgt ccattgccggtctctgtcttaattagcatctactccaagaagcttccctggtgagagctga gtggtccactgatgtacagttacagcattctttcaccagttcccatgctcatcagggacaa tagcctctggacttttaccacttcacatttctggtttctttctttgtattgttaatctacc gtcttaaaccaggctgttgtattctgggcaggatattttaagtccaactttctatcttcca tcttgaagtcctcttccaaggtactagtgttctaaaatgctactatgtttacatgactgca ctgtagaaagcctttccaatcaactttcagttacatgaagaggtgataaagtgccaggctg tgcagcatcatggaaatcttgtcatacgctagatggtgcttctcttttaaatttgtcatcc tttccctacatctagattacaccattttcgaacaataatggtttcctacacattttgttat gttgttccctatattttagaccatccccttgctgttttccattacctatgacactgccaaa atcacttttaaaaaattcagttcaaataactccctcaagagtcttaggactccatagcata tgtagtactgaccactctgtgtcttgtgctttttgtgtcttctgcagagaggttgacactc tttgttctacttggttattgcacagcttgattctctcagatggactgtgatctctgtgggt cagtgctgtgttggacacttctttgcatcctcaggattcagttcccaatgcttaatataaa ctcagagctcaacaaaaagtttttgagttatacatctgttcttatgtatagctctatagtc tgtgatccgttgagtccacttttcttcacacccttctccttatttggagtacttggggaga ttgtacagtgtaatggtagagaaagccaaaattgcctgaattaaaatcctactttgaacat tctttatttgtgtgagcacgtgaacctttgttaatttacctacagcttttgtgccttagta tacatacacacatatatacacacacatgcatacacacacacacacacacacacacacacac acacacacacacacacaatcagtaactctttgtgagtgttgttatgatgactgttctgaca tacaaacatgcatagaaagtgtccagaattagcattgtcaaccatcatttttcataattac ttcggttcatttattaacacttgcttttcctattagctgctcagtgctggttttcagagga caagtaagctggatgattatttccagagttggatcaaacaaaaatcctaaaccctcaagtc tccagaaacaattaaaatccaaccaatgttaacatatacagtcttgctttgaagaaccttc attctgctttctacataatcaaactgtataggtcccagattttggttagttcttcagttaa acatgtgctcttgcttaagaattttccattcttcaaaggattgtcaagctttcctgtggct ttctaaagttggaaaagtacaaatataatcttgtctatgcctgtcaaattgctgggtctga tcaggtcatagatggagagcaataaaattgtatcagaagagtatcaaaggaatgatgggcc tatgggcatttcatttccatatatttcttacttaatgaagaacattctagaaggttagcat ctgagctaagttcaagcctcgaatttcaaaagcctgaatatccttctacatggaaacacca tgggttttggaagcagacttcttatgaaagccgtcacacacacacacacacacacacacac acacacacacacacacacacaggtaaaatttgcataatgtaacgtcatgccagcagaaaag cctttcttaagttatttttttacttttataaaatacaaagcacttaaattcatagttactt aaagacggagggtaaccttccaatagcagacttgagattcaaaattagacgacggctgttt
tgttgcaagaaaaaaaaataataaccctgatgtattgttcccctccatctcttcccaaaga gctactcgacctacgaaacaaacaaatctcatcaggagcacagataaccccaagcttcaga cgtgtaatctgactgtggccatcagcaaccagaaatgagttttttctaatcagctttccat cagtcctcagtcactcatataaaggaacacggggaggggaggaagcgcactgctcttcagc accagggttctggacagcgccccgagcaggcagctgccactgcagttcctccttcatctct gccaaggccgcccctctggtcccccctgctgctgagaaagaaaattacatcgggagccatg gtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcg acgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaa gctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtg accaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacg acttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaagga cgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgc atcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagt acaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggt gaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccag cagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcaccc agtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgt gaccgccgccgggatcactctcggcatggacgagctgtacaagtaaagcggccctagagct cgctgatcagcctcgactgtgcctctagttgccagccatctgttgtttgcccctcccccgt gccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaatt gcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagca agggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttc tgaggcggaaagaaccagctggggctcgacgacgaagttcctatactttctagagaatagg aacttcctcgacggtatcgagcttctgatggaattagaacttggcaaaacaatactgagaa tgaagtgtatgtggaacagaggctgctgatctegttcttcaggctatgaaactgacaca11 tggaaaccacagtacttagaaccacaaagtgggaatcaagagaaaaacaatgatcccacga gagatctatagatctatagatcatgagtgggaggaatgagctggcccttaattggttttgc ttgtttaaattatgatatccaactatgaaacattatcataaagcaatagtaaagagccttc agtaaagagcaggcatttattctaatcccaccccacccccacccccgtagctccaatcctt ccattcaaaatgtaggtactctgttctcacccttcttaacaaagtatgacaggaaaaactt ccattttagtggacatctttattgtttaatagatcatcaatttctgcagacttacagcgga tcccctcagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcgg cgataccgtaaagcacgaggaagcggtcagcccattcgccgccaagctcttcagcaatatc acgggtagccaacgctatgtcctgatagcggtccgccacacccagccggccacagtcgatg aatccagaaaagcggccattttccaccatgatattcggcaagcaggcatcgccatgggtca cgacgagatcctcgccgtcgggcatgcgcgccttgagcctggcgaacagttcggctggcgc gagcccctgatgctcttcgtccagatcatcctgatcgacaagaccggcttccatccgagta cgtgctcgctcgatgcgatgtttcgcttggtggtcgaatgggcaggtagccggatcaagcg tatgcagccgccgcattgcatcagccatgatggatactttctcggcaggagcaaggtgaga tgacaggagatcctgccccggcacttcgcccaatagcagccagtcccttcccgcttcagtg acaacgtcgagcacagctgcgcaaggaacgcccgtcgtggccagccacgatagccgcgctg cctcgtcctgcagttcattcagggcaccggacaggtcggtcttgacaaaaagaaccgggcg cccctgcgctgacagccggaacacggcggcatcagagcagccgattgtctgttgtgcccag tcatagccgaatagcctctccacccaagcggccggagaacctgcgtgcaatccatcttgtt caatggccgatcccatattggctgcaggtcgaaaggcccggagatgaggaagaggagaaca gcgcgcagacgtgcgcttttgaagcgtgcagaatgccgggcctccggaggaccttcgggcg cccgcccgcccctgagcccgcccctgagcccgcccccggacccaccccttcccagcctctg agcccagaaagcgaaggagcaaagctgctattggccgctgccccaaaggcctacccgcttc cattgctcagcggtgctgtccatctgcacgagactagtgagacgtgctacttccatttgtc acgtectgcacgacgcgagctgeggggegggggggaacttectgactaggggaggagtaga
aggtggcgcgaaggggccaccaaagaacggagccggttggcgcctaccggtggatgtggaa tgtgtgcgaggccagaggccacttgtgtagcgccaagtgcccagcggggctgctaaagcgc atgctccagactgccttgggaaaagcgcctcccctacccggtagaattggccgcgaagttc ctatactttctagagaataggaacttcggccgccaccgcctcgatcgagtatcgagtatct ggcagtctccggtgttctctagggcatcaatggtcttaacctcaccatatggacacctgct gtagagtaagcatgaagttcaactcccttttcctcagtcgtcttgtcttactgcctccttt ctgtctgcctcagctctccacaaagtacaagcagctgggcgagttgctgggagataaactc tcctcaaatagaaagtgaagttccctatcgtgaagaatgaaagattatgtgaaccaatgtg ctaaattcactcctccttcaaagtatagaacttgaaaggctgataaggaagatttgggaga aagaattaaatacatctctctggtttctacaaggcaatattgtctcctatgccctacagag ctaatatgtgtattcttgagattaagtgcaggttttggaataaaagaggaaccagggccct tcaaaggtttccctgcatgccacattactctccctttaaagcttaattgatggaagagtta aggaagcattctgtttgcttcaatatttacaagatgtgtgtgtgtgtgtgtgtgtgtgtgt gtgtgtgtgtttaaaatactaacattaaagaatggtatttgaagagttacagttcttgcaa aggtttaggtcttaatttgaccaacagttatgagctttaataaacacagtggtttgatttg ctaacatgcaataaaatacacatccaaggttctgtcattaaaattttaagggatgcttcac cgttgtccttcctgtttatcaaccaattgcaaattcagctcgtcagagaaaaattgggtta atgccatagctccaagtatgtccattctatttattacccacaaacgctgaaaaacagtcac agcaactttcttagcactatttaagtcgatttgcgaaaatgaggccaaaatgtaaaatagc ttttctaatactcaagtatcatgaatatttgtatgaggtgttacattgaggaattccgaag ctgtaataaacggcaagaagaaaaccacgaagcccttcagttatggttaatgtatgacact ttcccataaaaatgcaccttagacatatgagacacaggcattacctggagaggggggagca gatgttgggaataggaagacatttgagcttctaatggctaaatatactgtcaagtaccagt gccagcataaactccttctctccctcaactcataaataataaaatatctattcctattata tataaactcggtgagggaggttatcttaatgcatggaaataaagccagtagtgaaatagga ggaagacaatggtaaatcacttaccactgggttccatgtaaatgtgcaggtcttagttcca cgaggatgtagtagactggtatgactatggtacctcaccattagccaggaagacagatctc tgtttgcccaactctgcaccataatcttacattgtctgtcaaatgggacattcctaggttt gttttttcaagttctgcagtgtgctctgatttttgctagactctgttattcctatccttag ctggaagaacttagtcattactcagattgtcgtgtgtgcccctaatggttagctagctggt tttctttctctctcatgcctttccaactgcctgtcctatatgtggctgaaacgtagtagtc actttccagactcaactgtccacggagggattcactgggcacgctgctctgtcactgactg cctcagtgagcaggaagattcagacttgggtccaaaggcaactccactttgagttggtatg atgggtggggcttgcagcttttaagaggaatctctggataaattgaatcattgaaacctct ggggtagaactgactcttgtgcttctactttcttgaagcaaaataaggctatcaaaagcga ggacaagaaaagcggcaaagagcccggcaaaggcgacaccacagagagcagcaagacagcg gtagtgttctccttgaagaatgaagttggtgggctggtgaaagcacttagactattccagg taaacacagagcactcgttcttctgtatcctcaaggtggctatgtgcctggtgatagactt gttccctgtcattaaggcatttctgggtaacatggcaaaagagtcaaaggaaccaaatgtc acaaagctttaaaaagggagtttactctgcccaacagtgaaataagcaggtttatgtgagc tgtgatttattaattcagactcagccaatagcaactctgttacttgttgccagatgaagtc tgtaattcaaaaatgtgagctttaaagtagaaaatgcaacacacacacatacatacaatac acacatacatatagacaaaacacacatacacagaacacacacatacacatagacaaaacac atacacagaacacacacatacacacacaatgcatatacacaaaacacacatacctatatac atacacacacaacacacaacacacatatacatacatatacatatacacatatacagaatac acacatatacacaatatacacatacacatagacaaaacacacatacacagaacacacacat acccaacagacatatacacacacaacgcacatacaaaacacacatacctacacacacatat acacacaacacactcacaacacacatatacatgcatacacacatacacacgtatatataca acacacatatacacatagacaaagcacacatacacaaaacacacatacctacagacacata cacacacaacacacatatacttatacacacacagacacacatatgcacacacacacacagt
gcttcaaaatgccaagagtctttatcctgctttgatttactaaactttaacagtacacatg tcatgagagcatatttgttgcataaaatgagatatatgagcacaggcacttattctcgtaa gcctcatttcatgaacgtaatcgaaggagtgtcttatagaaagatatgccacggtccctgg cacgagtccttgttttcagtcctaaatgaagagcttgggtgaggaggaacaccttgtccac ctggtgtcttctccacctgaagcaaaaaaccctcttacaaactacatgtggttgggagcga tggctggtcaccagggctgtaagataaacagttcaactctttctggtgccttaggctgctg tgtagcagacacatagttaggggatttggacctgtgttctgctgggttggctaactgctct gtgcacctcggtttgatttcctcatttgtaaaatggcaatgataccaattcctacctaact ggcttgtttggatgaggcgattttgtaaaatgcagacaggaggcattatttgtgatgcctc tagatgaccatagattgggaattaacctgactcggaaaatgtttccttgttcccctttggc cttgaagaaaggaccaggcaccataaaccgtgttaatggaaatctagagtagtattttaca cataattatgccatgtagggctgtgtccacatggtgcccgaaggcaagccaattcaatttg acagctgggtgcagtgggaaacatccctggggaaggggcttgcactttgaatttgcactct gccatcagctattcaccaggtctcagctaaaccacttaacccattgagatctcagtgccca gtctaaaaatacttactctgattcaaccaaaccttaaaagcccaaaatggcacaatgtaca tggagtctcttgggccttgaacttaatatcgat
As the PGK-neo cassette contains a prokaryotic promoter, in addition to the eukaryotic one, bacteria harbouring the recombinant vector were allowed to growth on a kanamycin selective agar plates. Screening of recombinant clones by means of colony- lifting assay and both restriction enzyme and sequencing analyses allowed to isolate a clone in which a homologous recombination event had occurred, generating the targeting vector Tph2eGFp(neo') suitable for homologous recombination in ES cells and containing left (SEQ ID No. 10) and right (SEQ ID No. 11) homology arm, 3.3 kb and 3.9 kb in size respectively.
SEQ ID No. 10: first or left homology arm
ggatcctcgagtataaattgcttctcagcaactttattgataactcggctttcttatgtct gcaaagtctttgatcattcttttatatcccgttaagcttctagtttttgttgctgatactt gtgaacttatattatttttgtatgcctcaaaaagaaaaggaggccttttatgtttttccat attttttaagtgaacaaaagtggttacagtattgaatagactcattaaatgccttaaatta actttaacattatttaaagcattttatatagtattcttatataatttattttgttcatatt tttctcctttcccaattcctcgcatgtccttaccacttcctcatctaccctattttatctt ctcttcctctcactcttcctcaaaaacataccccaaactgaaaacaaaaagagagcaaaac aaaaaataaagatcagaacaaatgatataaaaacattaggacaaaaaataccaaaactaaa taaaaagcacacagacacacccacacagacacacacccacacagacacacacacacacaca cacacacacacacacacacacacacacacacacagggagttcattttatgttggccaactg cttctgggcatggagcctgccctaaagtatgattgatgtacccaatgatgtaaacagattt ttcctttcttagaaagtatcacttgcaaatagcttcttggctagggataggactttgtgtc catttcctctcactggtctgaaatgtttgtctggtttggatttgtgcatgctttcgcagtc tctgtgagtttatatgtgcagtggacttgcagtgcttggataaaagtccttggagacatcc cccacctgtagctcagacactcttcctgctttctcagcattgataccttgaaggaaaatgt ttgataaagccatttcatttagtgatgactgttccagagtccctcattctctgctcattgt ccattgccggtctctgtcttaattagcatctactccaagaagcttccctggtgagagctga gtggtccactgatgtacagttacagcattctttcaccagttcccatgctcatcagggacaa tagcctctggacttttaccacttcacatttctggtttctttctttgtattgttaatctacc gtcttaaaccaggctgttgtattctgggcaggatattttaagtccaactttctatcttcca
tcttgaagtcctcttccaaggtactagtgttctaaaatgctactatgtttacatgactgca ctgtagaaagcctttccaatcaactttcagttacatgaagaggtgataaagtgccaggctg tgcagcatcatggaaatcttgtcatacgctagatggtgcttctcttttaaatttgtcatcc tttccctacatctagattacaccattttcgaacaataatggtttcctacacattttgttat gttgttccctatattttagaccatccccttgctgttttccattacctatgacactgccaaa atcacttttaaaaaattcagttcaaataactccctcaagagtcttaggactccatagcata tgtagtactgaccactctgtgtcttgtgctttttgtgtcttctgcagagaggttgacactc tttgttctacttggttattgcacagcttgattctctcagatggactgtgatctctgtgggt cagtgctgtgttggacacttctttgcatcctcaggattcagttcccaatgcttaatataaa ctcagagctcaacaaaaagtttttgagttatacatctgttcttatgtatagctctatagtc tgtgatccgttgagtccacttttcttcacacccttctccttatttggagtacttggggaga ttgtacagtgtaatggtagagaaagccaaaattgcctgaattaaaatcctactttgaacat tctttatttgtgtgagcacgtgaacctttgttaatttacctacagcttttgtgccttagta tacatacacacatatatacacacacatgcatacacacacacacacacacacacacacacac acacacacacacacacaatcagtaactctttgtgagtgttgttatgatgactgttctgaca tacaaacatgcatagaaagtgtccagaattagcattgtcaaccatcatttttcataattac ttcggttcatttattaacacttgcttttcctattagctgctcagtgctggttttcagagga caagtaagctggatgattatttccagagttggatcaaacaaaaatcctaaaccctcaagtc tccagaaacaattaaaatccaaccaatgttaacatatacagtcttgctttgaagaaccttc attctgctttctacataatcaaactgtataggtcccagattttggttagttcttcagttaa acatgtgctcttgcttaagaattttccattcttcaaaggattgtcaagctttcctgtggct ttctaaagttggaaaagtacaaatataatcttgtctatgcctgtcaaattgctgggtctga tcaggtcatagatggagagcaataaaattgtatcagaagagtatcaaaggaatgatgggcc tatgggcatttcatttccatatatttcttacttaatgaagaacattctagaaggttagcat ctgagctaagttcaagcctcgaatttcaaaagcctgaatatccttctacatggaaacacca tgggttttggaagcagacttcttatgaaagccgtcacacacacacacacacacacacacac acacacacacacacacacacaggtaaaatttgcataatgtaacgtcatgccagcagaaaag cctttcttaagttatttttttacttttataaaatacaaagcacttaaattcatagttactt aaagacggagggtaaccttccaatagcagacttgagattcaaaattagacgacggctgttt tgttgcaagaaaaaaaaataataaccctgatgtattgttcccctccatctcttcccaaaga gctactcgacctacgaaacaaacaaatctcatcaggagcacagataaccccaagcttcaga cgtgtaatctgactgtggccatcagcaaccagaaatgagttttttctaatcagctttccat cagtcctcagtcactcatataaaggaacacggggaggggaggaagcgcactgctcttcagc accagggttctggacagcgccccgagcaggcagctgccactgcagttcctccttcatctct gccaaggccgcccctctggtcccccctgctgctgagaaagaaaattacatcgggagcc
SEQ ID No. 11 : second or right homology arm
tcgagtatcgagtatctggcagtctccggtgttctctagggcatcaatggtcttaacctca ccatatggacacctgctgtagagtaagcatgaagttcaactcccttttcctcagtcgtctt gtcttactgcctcctttctgtctgcctcagctctccacaaagtacaagcagctgggcgagt tgctgggagataaactctcctcaaatagaaagtgaagttccctatcgtgaagaatgaaaga ttatgtgaaccaatgtgctaaattcactcctccttcaaagtatagaacttgaaaggctgat aaggaagatttgggagaaagaattaaatacatctctctggtttctacaaggcaatattgtc tcctatgccctacagagctaatatgtgtattcttgagattaagtgcaggttttggaataaa agaggaaccagggcccttcaaaggtttccctgcatgccacattactctccctttaaagctt aattgatggaagagttaaggaagcattctgtttgcttcaatatttacaagatgtgtgtgtg tgtgtgtgtgtgtgtgtgtgtgtgtgtttaaaatactaacattaaagaatggtatttgaag agttacagttcttgcaaaggtttaggtcttaatttgaccaacagttatgagctttaataaa cacagtggtttgatttgctaacatgcaataaaatacacatccaaggttctgtcattaaaat
tttaagggatgcttcaccgttgtccttcctgtttatcaaccaattgcaaattcagctcgtc agagaaaaattgggttaatgccatagctccaagtatgtccattctatttattacccacaaa cgctgaaaaacagtcacagcaactttcttagcactatttaagtcgatttgcgaaaatgagg ccaaaatgtaaaatagcttttctaatactcaagtatcatgaatatttgtatgaggtgttac attgaggaattccgaagctgtaataaacggcaagaagaaaaccacgaagcccttcagttat ggttaatgtatgacactttcccataaaaatgcaccttagacatatgagacacaggcattac ctggagaggggggagcagatgttgggaataggaagacatttgagcttctaatggctaaata tactgtcaagtaccagtgccagcataaactccttctctccctcaactcataaataataaaa tatctattcctattatatataaactcggtgagggaggttatcttaatgcatggaaataaag ccagtagtgaaataggaggaagacaatggtaaatcacttaccactgggttccatgtaaatg tgcaggtcttagttccacgaggatgtagtagactggtatgactatggtacctcaccattag ccaggaagacagatctctgtttgcccaactctgcaccataatcttacattgtctgtcaaat gggacattcctaggtttgttttttcaagttctgcagtgtgctctgatttttgctagactct gttattcctatccttagctggaagaacttagtcattactcagattgtcgtgtgtgccccta atggttagctagctggttttctttctctctcatgcctttccaactgcctgtcctatatgtg gctgaaacgtagtagtcactttccagactcaactgtccacggagggattcactgggcacgc tgctctgtcactgactgcctcagtgagcaggaagattcagacttgggtccaaaggcaactc cactttgagttggtatgatgggtggggcttgcagcttttaagaggaatctctggataaatt gaatcattgaaacctctggggtagaactgactcttgtgcttctactttcttgaagcaaaat aaggctatcaaaagcgaggacaagaaaagcggcaaagagcccggcaaaggcgacaccacag agagcagcaagacagcggtagtgttctccttgaagaatgaagttggtgggctggtgaaagc acttagactattccaggtaaacacagagcactcgttcttctgtatcctcaaggtggctatg tgcctggtgatagacttgttccctgtcattaaggcatttctgggtaacatggcaaaagagt caaaggaaccaaatgtcacaaagctttaaaaagggagtttactctgcccaacagtgaaata agcaggtttatgtgagctgtgatttattaattcagactcagccaatagcaactctgttact tgttgccagatgaagtctgtaattcaaaaatgtgagctttaaagtagaaaatgcaacacac acacatacatacaatacacacatacatatagacaaaacacacatacacagaacacacacat acacatagacaaaacacatacacagaacacacacatacacacacaatgcatatacacaaaa cacacatacctatatacatacacacacaacacacaacacacatatacatacatatacatat acacatatacagaatacacacatatacacaatatacacatacacatagacaaaacacacat acacagaacacacacatacccaacagacatatacacacacaacgcacatacaaaacacaca tacctacacacacatatacacacaacacactcacaacacacatatacatgcatacacacat acacacgtatatatacaacacacatatacacatagacaaagcacacatacacaaaacacac atacctacagacacatacacacacaacacacatatacttatacacacacagacacacatat gcacacacacacacagtgcttcaaaatgccaagagtctttatcctgctttgatttactaaa ctttaacagtacacatgtcatgagagcatatttgttgcataaaatgagatatatgagcaca ggcacttattctcgtaagcctcatttcatgaacgtaatcgaaggagtgtcttatagaaaga tatgccacggtccctggcacgagtccttgttttcagtcctaaatgaagagcttgggtgagg aggaacaccttgtccacctggtgtcttctccacctgaagcaaaaaaccctcttacaaacta catgtggttgggagcgatggctggtcaccagggctgtaagataaacagttcaactctttct ggtgccttaggctgctgtgtagcagacacatagttaggggatttggacctgtgttctgctg ggttggctaactgctctgtgcacctcggtttgatttcctcatttgtaaaatggcaatgata ccaattcctacctaactggcttgtttggatgaggcgattttgtaaaatgcagacaggaggc attatttgtgatgcctctagatgaccatagattgggaattaacctgactcggaaaatgttt ccttgttcccctttggccttgaagaaaggaccaggcaccataaaccgtgttaatggaaatc tagagtagtattttacacataattatgccatgtagggctgtgtccacatggtgcccgaagg caagccaattcaatttgacagctgggtgcagtgggaaacatccctggggaaggggcttgca ctttgaatttgcactctgccatcagctattcaccaggtctcagctaaaccacttaacccat tgagatctcagtgcccagtctaaaaatacttactctgattcaaccaaaccttaaaagccca aaatggcacaatgtacatggagtctcttgggccttgaacttaatatcgat
Homologous recombination in ES cells
Electroporation of Tph2eGFP(neo> targeting vector in ES cells and identification of recombinant clones
Electroporation is the preferred method to introduce the targeting vector within ES cells as DNA concentration and cell density can be adjusted to favour vector integration in single- copy. Any commercially available ES cells may be used. The cells line were maintained on gelatinized tissue culture dishes in ES cell medium containing 1000 U/ml leukemia inhibitory factor (LIF) according to BayGenomics protocols available online <http ://baygenomics.ucsf.edu/>.
The Tph2eGFP(neo) targeting vector was purified on a Qiagen column and 2 x 107 ES cells/ 0.7 ml of PBS cells trypsinized from 80% confluent plates were added of 30 μg of linearized targeting vector and electroporated using a Bio-Rad Gene Pulser unit. Electroporated ES cells were transferred in 40 ml of ES cell medium and plated to a confluence of 5 x 106 cells per tissue-culture petri dishes 10 cm diameter. After G418 selection, cells that have integrated the targeting vector form colonies about 1 mm in size. Recombinant resistant colonies were picked and replicated in 96-well plates. Clones contained in one replica-plate were stocked at -80°C while clones contained in the second set of plates were used for Southern blot analysis. Genomic DNA was extracted from single ES cell clones, digested with specific restriction enzymes and separated according to size by gel-electrophoresis. In particular, genomic DNA was digested with Bgll and a probe upstream the left homology arm (Probe A) was used to screen for proper 5' recombination. Expected size of signal on autoradiography was 12,5 kb for wild-type allele and 7,3 kb for the recombinant one. Stul digestion and probe B were used to screen for the 3' correct recombination to obtain 8 kb and 5.6 kb for wild type and recombinant allele, respectively. Positive ES cell clones in which a correct homologous recombination event had occurred were identified (Figure le). In the recombinant Tph2eGFP^neo-> ES cell clones, 105 nucleotides of exon 1 downstream the first ATG codon encoding 35 amino acids of the enzyme Tryptophan hydroxylase 2 were replaced with the cDNA for the vital reporter gene eGFP and the neo cassette flanked by FRT sites {Tph2eGFP(neo^ knock-in ES cell clones).
Generation of Tph2elJ^ mouse ES cell
Flp mediated recombination in the Tph2eGFP(neo) knockin ES cell
According to previous observations (Pasqualetti et al, 2002), the presence of the PGK-neo cassette is expected to interfere with the normal Tph2 regulation and to prevent eGFP reporter gene expression in differentiating Tph2eGFP^neo-> knock- in ES cells. To circumvent this possibility and to ensure that the newly generated ES cell works as an in vitro sensor for serotonergic neuron differentiation, Flp recombinase was transiently expressed in Tph2eGFp("eo) ES cells in order to induce Flp-mediated excision of the PGK-neo cassette in the genome.
Briefly, the procedure was the following: Tph2eGFP(neo) ES cells were transfected with the pCAGGS-FLPe vector (Gene Bridges GmbH, Cat. No. A201) that expresses Flp recombinase and confers resistance to puromycin. The day of the electroporation, 12xl06 of 80% confluent cells were electroporated in presence of 15μg of the pCAGGS-FLPe vector. The following day the culture medium was replaced for 48 hours with medium containing l^g/ml puromycin to select transfected cells (see also: Schaft et al, 2001). Once colonies were formed and reached about 1 mm in size, 200 recombinant resistant colonies were picked and were replicated in 96-well plates. PCR analysis on genomic DNA extracted from each clone allowed to identify 3 clones in which the PGK-neo cassette had been excised to obtain the new allele called Tph2eGFP . Finally, the region flanking the FRT site was sequenced to confirm that Flp-mediated recombination occurred correctly in the three clones.
Tph2eGFP ES cells maintained totipotency
As it is known that long-term culture and in vitro handling could affect ES cell karyotype or multi-lineage differentiation capacities, metaphase cells were analyzed in order to verify the correct karyotype. Subsequently, to verify that Tph2eGFP ES cells maintained a totipotent state, the expression of stem cell molecular markers such as Oct4 was checked. Alkaline phosphatase (AP) activity assay, that is commonly used to evaluate maintenance and self-renewal of embryonic stem cells, was also carried out. Tph2eGFP ES cells showed high levels of Oct4 expression (Fig. 2a) and strong AP staining (Fig. 2b) indicating that they maintained totipotency.
Tph2eGFP ES cell differentiation toward serotonergic neurons
In the last few years, several protocols have been described to obtain neuronal
differentiation in vitro from ES cells. The strategies can be grouped in two main approaches: the first approach is mimicking the environment that produces neuroectoderm in the embryo by providing appropriate cell-cell interactions and signals through formation of aggregates made up of cells of all the three primary germ layers (embryoid body, EBs); the second approach is to deprive the ES cells of both cell-cell interactions and signals by low density culture in serum-free medium, evoking a default mechanism for neural differentiation.
In the present invention, several protocols were used to differentiate Tph2eGFP ES cell toward serotonergic neurons in vitro. Neuronal differentiation of ES cells is based on conventional protocols known to those skilled in the art. Two approaches present in literature (Lee et al, 2000; Fico et al, 2008) were used. The authors successfully obtained in vitro serotonergic neurons expressing eGFP. This demonstrates the versatility of the Tph2eGFP ES cell in differentiating toward serotonergic neural phenotype.
In order to assess the nature of eGFP expressing neurons, double immunocytochemistry experiment using specific antibodies for serotonin and eGFP was performed. Immunocytochemistry was carried out using standard protocols and antibodies were used as follows: rabbit anti-5-HT (Sigma Cat. No. s 5545) 1 :500, chicken anti-eGFP (Aves Labs Cat. No. GFP-1020) 1 :200; Rhodamine Red-X goat anti-rabbit IgG, Fluorescein (FITC) labeled anti-chicken IgY. Secondary antibody dilutions were: Rhodamine Red-X goat anti- rabbit IgG (Invitrogen Cat. No. R-6394) 1 :500; Fluorescein (FITC) labelled anti-chicken IgY (Aves Labs Cat. No. F-1005) 1 :500. Results showed co-localization of serotonin and eGFP demonstrating that eGFP expressing cells are in vitro differentiated serotonergic neurons (Fig. 3 a, b). Moreover, by means of RT-PCR analysis on differentiated ES cells, expression of specific serotonergic markers such as Tph2, serotonin transporter (SERT) and serotonin receptor 1A was detected (Fig. 3c), thus allowing to further conclude that appearance of eGFP correlates with the activation of a specific program for serotonergic neuron differentiation. Taken together, synthesis of serotonin and activation of specific molecular markers suggest that serotonergic neurons differentiated from Tph2eGFP ES cells in vitro. Thus, such cells provide a suitable model system for the study of a variety of aspects related to development, biology and functionality of serotonergic neurons. Serotonergic neurons differentiated in vitro appear as multipolar neurons and exhibit long projections forming distinct axonal fascicles and synapses. Morphologically they resemble serotonergic neurons present in the raphe nuclei of the mouse brain.
REFERENCES
Copeland et al (2001). Nat Rev Genet. 2, 769-779.
Dobie et al (1997). Trends Genet. 13(4): 127-30.
Fico, A., et al. (2008). Stem Cells Dev. 17, 573-84.
Garrick et al (1998). Nat Genet. 18(l):56-9
Lee, S.H., et al. (2000). Nat Biotechnol. 18, 675-9.
Pasqualetti et al. (2002). Genesis 32, 109-111.
Rijli et al (1993). Cell 75: 1333-1349.
Rodriguez et al (2000). Nat Genet 25: 139-140
Schaft, J., et al. (2001). Genesis 31, 6-10.
Yadav et al (2009). Cell 138(5):976-89
Yu, D., et al. (2000). Proc Natl Acad Sci U S A. 23, 5978-5983.
Claims
1- A stem cell able to express an exogenous vital reporter coding sequence upon differentiation into serotonergic neurons wherein the exogenous vital reporter gene coding sequence encodes for a fluorescent protein.
2- The stem cell of claim 1 wherein the fluorescent protein is selected from the group of: enhanced green fluorescent protein (eGFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP).
3- The stem cell of claim 1 or 2 being an adult stem cell.
4- The stem cell of claim 3 being a murine or a human adult stem cell.
5- The stem cell of claim 1 or 2 being a non-human mammalian embryonic stem cell.
6- The stem cell of any of previous claims being an induced pluripotent stem cell (iPS cell).
7- The stem cell of any one of previous claim wherein the exogenous vital reporter coding sequence is under the control of a promoter and/or a regulating sequence of the Tph2 gene, encoding for a Tph2 protein of SEQ ID No. 12, or allelic variants thereof.
8- A method to generate the stem cell of any one of previous claims comprising the step of inserting the exogenous vital reporter coding sequence into the stem cell genome so that it is under the control of a promoter and/or a regulating sequence of the Tph2 gene, encoding for a Tph2 protein of SEQ ID No. 12, or allelic variants thereof.
9- The method of claim 8, wherein the insertion of the exogenous reporter coding sequence is performed by homologous recombination so that the initiation methionine codon of the Tph2 coding sequence results to be in frame with the reporter gene coding sequence.
10- The method according to claim 9 wherein the homologous recombination is performed by means of a recombinant vector comprising from the 5 ' to the 3 ' end direction:
- a first homology nucleotide sequence arm comprising SEQ ID No. 10;
- a sequence wherein the initiation methionine codon of the Tph2 coding sequence is in frame with the reporter coding sequence;
-a selectable cassette sequence that is removable through the action of site specific recombinase, and
- a second homology nucleotide sequence arm comprising SEQ ID No. 11.
11- The stem cell of any one of claim 1 to 7 for medical use.
12- Use of the stem cell of any one of claim 1 to 7 to monitor serotonergic neuron
development and/or function.
13- The use of claim 12 wherein the serotonergic development and/or function is monitored by measuring and/or assessing a parameter selected from the group of: cellular morphology, neurite growth, neurite elongation and/or degeneration, neuronal cell death, neurogenesis, neuronal electrical properties, synaptogenesis or expression of molecular serotonergic markers.
14- A method to identify a molecule able to induce or to inhibit the differentiation of stem cells into serotonergic neuron comprising the steps of:
a) providing the stem cell of any one of claims 1 to 7;
b) exposing said cell to a compound library comprising putative inducing or inhibiting differentiation agents;
c) detecting and/or measuring the fluorescence.
15- The method of claim 14 being performed by High Throughput Screening.
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CN113493805A (en) * | 2021-07-06 | 2021-10-12 | 上海市东方医院(同济大学附属东方医院) | Construction method and application of human serotonin neuron TPH2 reporter gene cell line |
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