CN1748034A - Improved genetic elements providing high levels of expression - Google Patents
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Abstract
The invention relates to improved genetic elements providing high levels of expression of operably-linked genes in a variety of tissues. In particular, fragments of unmethylated, CpG islands of less than 2kb are shown to provide enhanced transgene expression and have advantages in terms of vector construction and cloning capacity.
Description
Invention field
The present invention relates to contain the polynucleotide of the open element of improved, less omnipresence chromatin (UCOE).In the time can being operationally connected to effable nucleotide sequence, described element produces high and gene expression dose repeatably.The invention still further relates to the carrier that contains described polynucleotide sequence, contain the host cell and the application in treatment of described polynucleotide, carrier or host cell of this carrier, or be used for application at the cell cultures expressing protein.
Background of invention
Current higher eucaryote chromatin Structure model assumption gene organization is at " structural domain form " (Dillon, N.﹠amp; Grosveld, F. chromatin Structure territory is as the gene function unit of potential eucaryon, Curr.Opin.Genet.Dev.4,260-264 (1994); Higgs, the open chromatin Structure territory Cell 95 of D.R.LCRs, 299-302 (1998)).The chromatin Structure territory imagination with concentrate, " closure ", Transcriptional Silencing state, or go configuration existence spissated, " open " and transcriptional activity.Being characterized as DNasel susceptibility increases, and the foundation of DNA supermethylation and the super acetylizad open chromatin Structure of histone is considered to initial gene and expresses necessary.
Chromatin regional opening and the character of closing are reflected in random integration in the host cell gene group in the genetically modified behavior.When being incorporated into the different position of mouse genome, identical construction produce different tissue specificities and the etap specific expressed pattern (Palmiter, R.D.﹠amp; Brinster, R.L Ann.Ref.Genet.20,465-499 (1986); Allen, N.D.et al.Nature 333,852-855 (1988); Bonnerot, C., Grimber, G., Briand, P.﹠amp; Amp; Nicolas, J.F.Proc.Natl.Acad.Sci.USA 87:6331-6335 (1990)).
Also observe the piebald expression pattern in given genetically modified mouse tissue frequently, common name is pasition effect variegation (PEV) (Kioussis, D.﹠amp; Festenstein, R.Curr.Opin.Genet.Dev.7,614-619 (1997)).When the gene of external source was integrated in the mammalian cell karyomit(e) of vitro culture, many integration incidents caused that transgenosis is reticent rapidly, and remaining causes bigger change (Pikaart, M.J., Recillas-Targa, the F.﹠amp of expression level; Felsenfield, G.Genes Dev.12,2852-2862 (1998); Fussenegger M., Bailey, J.E., Hauser, H.﹠amp; Amp; Mueller, P.P Trends Biotech.17,35-42 (1999)).These position effect render transgenic expression efficiencies reduce, and show the application potential of its fundamental research and biotechnology.
The chromatin Structure domain model of gene organization shows that the Genetic Control element that can set up with the open chromatin Structure of keeping the energy transcriptional activity should be relevant with genomic active region.
Region territory (LCRs) is that a class has the transcriptional regulatory element that long-range chromatin is transformed ability.Transgenic mice LCRs by them on particularly single copy transgenosis on the gene that cis connects, integration site is ind, the ability of transgenosis copy number expression that rely on, physiological level and with the functional mode definition, Fraser, P.﹠amp; Grosveld, F.Curr.Opin.Cell Biol.10,361-365 (1998); Li, Q., Harju, S.﹠amp; Amp; Peterson, K.R.Trends Genet.15:403-408 (1999).Importantly, this expression is tissue-specific.LCRs can hinder heterochromatic distribution, prevents PEV (Kioussis, D.﹠amp; Festenstein, R.Curr.Opin.Genet.Dev.7,614-619 (1997)), and by a series of be positioned at 5 of gene that their regulate ' or 3 ' DNase I super quick (HS) site form (Li, Q., Harju, S.﹠amp; Peterson, K.R.Trends Genet.15:403-408 (1999)).
LCRs show as by two independent, although not necessarily independently component form.At first, set up " open chromatin Structure territory ", secondly, translate activation capacity significantly and produce the dependent expression of transgenosis copy number (Fraser, P.﹠amp; Grosveld, F.Curr.Opin.Cell Biol.10,361-365 (1998).The molecular mechanism that LCRs brings into play its function remain the question in dispute (Higgs, D.R.Cell 95,299-302 (1998); B μ lger, M.﹠amp; Groudine, M.GenesDev.13,2465-2477 (1999); Grosveld F.Curr.Opin.Genet Dev.9152-157 (1999); Bender, M.A., B μ lger, M., Close, J.﹠amp; Amp; Groudine, M..Mol.Cell 5,387-393 (2000).
The propagation of producing the mammal cell line of high level treatment protein product is industry in the main development.The chromatin position effect makes it to become process difficulty, not only time-consuming but also expensive.The method of this Mammals of the most normally used production " cell factory " depend on by drug resistance gene the gene amplification of combination inductive (for example, DHFR, glutamine synthetase (Kaufman RJ. Enzymology method 185, and keep strict selective pressure 537-566 (1990)).Contain carrier by utilization from the LCRs of cance high-expression gene structural domain, use is from the cell of suitable tissue, simplify described method widely, produce cloned cell line (the Needham M of the demonstration high level expression of vast scale, Gooding C, Hudson K, Antoniou M, Grosfeld F and Hollis M.Nucleic Acids Res 20,997-1003 (1992); Needham M, Egerton M, MillestA, Evans S, Popplewell M, Cerillo G, McPheat J, Monk A, Jack A, Johnstone D and Hollis M.Protein Expr Purif 6,124-131 (1995).
Yet the tissue specificity of LCRs although useful under some environment, remains the controlling factor of a lot of application, the known LCR of not having in the tissue that for example needs to express, or require in many or whole tissues, to express.
Be incorporated herein by reference herein we at careful patent application PCT/GB99/02357 (WO00/05393), US 09/358082, GB 0022995.5 and US 60/252,048, described the element of being responsible for setting up open chromatin Structure territory in their natural dyeing body environment, the site of only being made up of the house-keeping gene of omnipresence expression is crossed in described open chromatin Structure territory.These elements are not from LCR, and comprise the no methylated CpG island of extension.We use the open element of chromatin (UCOE) of term omnipresence to describe this element.
In mammalian DNA, dinucleotides CpG is by being that the dnmt rna of 5-methylcytosine is identified with cytosine methylation.Yet 5-methylcytosine is unsettled, and is converted into thymus pyrimidine.As a result, the probability much less that occurs at random of the generation frequency ratio of CpG dinucleotides.Yet some zones of genomic dna have the CpG that approaches to expect frequency, and these sequences are called as " CpG island "." the CpG island " of Shi Yonging is defined as the dna sequence dna of 200bp at least herein, its CpG content ratio with at least 50% GC content and observation/expectation is at least 0.6 (be CpG dinucleotides content for expected value at random at least 60%) (Gardiner-Green M and Frommer M.J Mol Biol 196,261-282 (1987); Rice P, Longden I and Bleasby A Trends Genet 16,276-277 (2000).
No methylated CpG island is known (Bird etc. (1985) the Cell 40:91-99 of prior art, Tazi and Bird (1990) Cell 60:909-920), and when the cytosine(Cyt) residue of remarkable ratio does not methylate, can be defined as the CpG island, and 5 ' end of its (0.1-3kb) Divergence that extends beyond two close space lengths usually gene of transcribing.These zones of DNA are reported in institute and keep supermethylation (Wise and Pravtcheva (1999) Genomics 60:258-271) in whole etap in a organized way.They are often relevant with 5 ' end of omnipresence expressing gene, and estimate 40% the restricted express spectra of gene display organization (Antequera, F.﹠amp; Bird, A.Proc.Natl.Acad.Sci.USA 90,1195-11999 (1993); Cross, S.H.﹠amp; Bird, A.P.Curr.Opin, Genet.Dev.5,309-314 (1995), and known be locating area (Tazi, the J.﹠amp of active chromatin; Bird, A.Cell, 60,909-920 (1990).
' extend ' no methylated CpG island be to cross over to comprise the zone that surpasses a transcription initiation site and/or cross over the no methylated CpG island that surpasses 300bp and the preferred 500bp of surpassing.The border on the no methylated CpG island of extending is by using PCR in this zone, and collaborative use has in the ability of recognition sequence digestion (cutting) DNA carries out functional specification to the restriction enzyme of the methylation state sensitivity of any CpG residue of appearance.Such enzyme is Hpall, the CCGG site that its identification and digestion exist in the CpG island usually, but only do not have to carry out under the methylated situation at the CG of central authorities residue.Therefore, with the DNA of Hpall digestion and comprise the PCR that carry out in the zone in Hpall site, if DNA is unmethylated then because Hpall digestion does not produce amplified production.PCR will be to produce amplified production under the methylated situation at DNA only.Therefore, methylate in the nothing of Hpall indigestion DNA that the zone is outer can observe pcr amplification product, thereby defined the border on " the no methylated CpG island of extension ".
We verified (WO 00/05393) across comprise from conjugated protein (the TBP)/proteoplast of people TATA component-B1 (PSMBI) and nuclear inequality-ribonucleoprotein A2/B1 (hnRNP A2)/heterochromatin albumen 1Hsy (HP1
Hs γ) zone on no methylated CpG island of allos bigeminy Divergence transcripting promoter of locus, produce repeatably, the genetic expression of physiological level, and can stop piebald expression pattern and the silence that usually occurs in transgenosis integration in the central heterochromatin.
No matter the term of Shi Yonging " is repeatably expressed " and is referred to polynucleotide of the present invention no matter its chromatin environment is directly expressed effable gene at identical basically expression level herein, preferably the cell type or the types of organization of polynucleotide of the present invention.Those skilled in the art will discern and obtain the expression of the expressible gene that is operatively connected of par basically, and no matter the polynucleotide chromatin environment of claim and preferred no matter cell type is supposed initiatively expressing gene of described cell.
We show that (WO 00/05393) no methylated CpG island relevant with the promotor of initiatively transcribing has the chromatinic ability of transformation, thereby and think to set up and keep the basic determinative in the open architecture territory of house-keeping gene seat.
UCOEs increases by level and the stable ratio of voluminous gene delivery incident that makes of improving transgene expression.This has important research and biotechnology applications, comprises the preparation transgenic animal and produce the recombinant protein product in cultured cells.We show that (WO 00/05393) UCOEs has the advantageous effects of the erythropoietin protein expression of pharmaceutical use to CMV-EGFP reporter gene construct and excretory.The character of UCOEs is further illustrated in the practicality in the gene therapy, and its validity often is restricted (Verma, I.M.﹠amp because low-frequency voluminous gene delivery and expression level and time length are insufficient; Somia, N.Nature389:239-242 (1997).
We above-mentioned application PCT/GB99/02357 (WO 00/05393) discloses ' 5.5RNP ' fragment (being disclosed in p11, the 6th and 7 row) of 4102 to 8286 Nucleotide definition of functional UCOE fragment, especially Figure 21 of about 4.0kb.Same application discloses ' 1.5kb RNP ' fragment (Figure 22 and 29, the source is described in p51, the 1st to 5 row).Yet this fragment is actually the BamHl-Tth111I fragment of above-mentioned ' 5.5RNP ' segmental 2165bp, is made up of 4102 to 6267 Nucleotide of this application.
In other application (WO 02/24930), we disclose the artificial constructed segmental UCOEs in naturally occurring CpG island that comprises.Disclosed fragment is bigger than the fragment that requires in the current application, and thinks and can not use little fragment separately at that time, and is not only the component as synthetic or ' blended ' UCOE construction.
Even there is the application of these significant hints and a wider range, the needs of further optimization transgene expression level are arranged still.The level that needs further preferred transgene expression is arranged, especially in vivo field of gene and produced in vitro recombinant protein field.
One need be the size that reduces the element that is used for reinforcing gene expression especially.So, littler carrier can be produced, or the carrier of bigger inset can be stably held and express.
The invention summary
An object of the present invention is to provide the open element of little chromatin to strengthen the genetically modified level and the repeatability that can be operatively connected.
According to the present invention, provide to comprise the segmental polynucleotide of little function UCOEs.This polynucleotide comprise that being no more than about 2kb does not have the methylated CpG island, or are no more than the fragment of about 2kb greater than this island.
Although comprising the big polynucleotide on the no methylated CpG island of prolongation is prior art known (referring to the application WO 00/05393 before the applicant oneself), did not determine whether to use obviously little fragment in the past and still kept expression level and the conforming enhancing of not having the acquisition of methylated CpG island by big UCOEs/.
The term of Shi Yonging " can be operatively connected " relation that refers to operability between the polynucleotide element of the present invention herein." can be operatively connected " is to well known to a person skilled in the art term, has described the functional relationship between the cis acting dna sequence dna.Structural relation is can yes or no related accurately, and different component type differences.As for promotor, its hint is positioned at the position with (usually less than 100bp within) of the open reading frame 5 of its driving ' adjacent basically.With regard to the no methylated CpG island of extending, it seems level and the consistence of the local action of chromatin Structure being responsible for improving genetic expression.For instance, the element that comprises the no methylated CpG island of extension is placed in and controls the next-door neighbour 5 ' end of the promotor that expressible gene transcribes.Yet " can be operatively connected " comprises the possibility that it is placed in elsewhere, as long as can show clear and definite functional effect.
The invention provides isolating polynucleotide, it comprises
A) the no methylated CpG island of Yan Shening;
B) effable open reading frame can be operationally connected to the no methylated CpG island of described extension;
C) promotor can be operationally connected to described open reading frame, and wherein said promotor is not connected to described CpG island natively;
It is characterized in that described CpG island size is no more than 2kb and wherein the repeated expression of the described open reading frame of acquisition in two types of organizations.
Perhaps, polynucleotide comprise that people hnRNP A2 gene is no more than the fragment of 2kb, preferably are no more than 1.6kb, comprise the Esp31 restriction fragment of 1546bp, the more preferably sequence of Fig. 2,977 to 2522 Nucleotide (SEQ ID NO:1), or its functional analogue.Preferably, described fragment is orientated forward.
' function homologue ' refer to can hybridize under stringent condition to the polynucleotide sequence of described disclosed sequence, and it has the character that effable open reading frame that similar realization can be operatively connected can repeat to express in two or more tissues.Strict hybridization/wash conditions is that prior art is known.For example, at 0.1 * SSC, 0.1%SDS stable nucleic acid hybridization thing after 60 ℃ of washings.If nucleotide sequence is known, the calculating optimum hybridization conditions is well known in the art.For example, hybridization conditions can decide according to nucleic acid GC content to be hybridized.Referring to (1989) such as Sambrook, molecular cloning; Laboratory method.The formula of the general calculation stringent condition that acquisition hybridization needs between the nucleic acid molecule of specifying similarity is:
T
m=81.5 ℃+16.6Log[Na+]+0.41[%G+C]-0.63 (% methane amide)
Preferred polynucleotide of the present invention promote that the non-tissue specificity of the gene ground that can be operatively connected can repeat to express.
In other preferred version, polynucleotide comprise that people hnRNP A2 gene is no more than the fragment of about 1kb, preferably include the BspE1-Esp31 restriction fragment of 987bp, more preferably comprise the sequence of Fig. 2,1536 to 2522 Nucleotide (SEQ ID NO:2).
Preferably, polynucleotide of the present invention comprise one or more naturally occurring promotors.
Preferably, polynucleotide of the present invention comprise the promotor that bigeminy or two-way Divergence are transcribed.In other embodiments, polynucleotide of the present invention comprise the fragment of beta-actin CpG island/promoter region, preferably come from the people.Polynucleotide more preferably of the present invention comprise the dna fragmentation of 100bp to the interior people of leap of 2kb scope beta-actin CpG island/promoter region.
In other optional embodiments, polynucleotide of the present invention comprise the fragment of PDCD2 CpG island/promoter region, preferably come from the people.More preferably, polynucleotide of the present invention comprise the dna fragmentation of 100bp to the interior people of leap of 2kb scope PDCD2 CpG island/promoter region.
Preferably, polynucleotide of the present invention comprise that 100bp crosses over the dna fragmentation of the dna fragmentation of people's beta-actin CpG island/promoter region and 100bp leap people PDCD2 CpG island/promoter region in the 2kb scope in the 1.9kb scope.Preferably, the promotor direct neighbor of described fragment and their Divergence direction.
Another aspect, the invention provides provides the carrier that comprises above-mentioned disclosed polynucleotide of the present invention.Preferably, described carrier is the expression vector that is used for eukaryotic gene expression through adjustment.
Usually described adjustment comprises, such as but not limited to, supply with the transcriptional control sequence (promoter sequence) of mediated cell/tissue specific expression.
Promotor and enhanser are terms well known in the art, and following provide for example rather than as the feature of restriction is provided.Promotor is 5 ', the regulating and controlling sequence of cis acting is directly connected to transcription initiation.Promoter element comprises so-called TATA box and is used to select the initial selection of RNA polymerase (RIS) sequence of transcription initiation position.These sequences are also in conjunction with being used for the polypeptide that RNA polymerase promotes that transcription initiation is selected.
Preferably, promotor is selected from CMV, EF-1 α, Rous sarcoma virus (RSV) LTR or HIV2 LTR or from its deutero-composite sequence.More preferably, promotor be CMV immediately/early promoter.Most preferably its be mouse CMV immediately/early promoter.
In the embodiment of carrier most preferably, CpG of the present invention island is positioned at the adjacent and 5 ' end of operation start with the expression of the effable open reading frame of control.
Enhancer element is the cis acting nucleotide sequence, often be found in genetic transcription initiation site 5 ' (and even enhanser can also be found in 3 of gene order ' be positioned at intron sequences and therefore be the position independently).The effect of enhanser is to improve the gene transcription ratio that is connected to enhanser.The enhanser activity is corresponding to the transcription factor (polypeptide) of trans-acting, and it has shown that specificity is attached on the enhancer element.Combination/the activity of transcription factor is corresponding to many factor of environmental, it comprise such as but not limited to, mesostate (for example glucose), environmental effect device (for example heating).(referring to the eukaryotic transcription factor, David S Latchman, Academic Press Ltd, SanDiego).
Adjustment also comprises supplies with selectable marker thing and autonomously replicating sequence, and the both promotes described carrier keeping in eukaryotic cell or prokaryotic hosts.The carrier of independently keeping is called episomal vector.So episomal vector is because their self-replacation and not need to integrate and can continue be desirable.This class episomal vector is described in W098/07876.
Promote the adjustment of the vector expression of encoding gene to comprise supply Transcription Termination/polyadenylic acid sequence.This also comprises supplies with internal ribosome entry site (IRES), and it acts on the genetic expression maximization that makes arranged carrier coding in two cis or many cis expression cassette.
These adjustment are that prior art is known.A large amount of documents of delivering about expression vector establishment and recombinant DNA method are arranged usually.See also Sambrook etc. (1989) molecular cloning: experiment guide, Cold Spring Harbour, NY and reference wherein; Marston, F (1987) dna clone technology: practical approach Vol III IRL Press, Oxford UK; DNA Cloning:FM Ausubel etc., the up-to-date handbook of molecular biology, John Wiley ﹠amp; Sons, Inc, (1994).
Described carrier can be episomal vector or integrating vector.Preferably, this carrier is a plasmid.Perhaps, carrier can be virus, for example adenovirus, adeno-associated virus (AAV), simplexvirus, vaccinia virus, slow virus or other retrovirus.
Preferred described carrier comprises the gene that can be operatively connected, and it is a therapeutic nucleic acids.This therapeutic nucleic acids can work for example cystic fibrosis of described disease, thalassemia, reaping hook anemia, FanconiShi anemia, hemophilia, Reconstruction in Sever Combined Immunodeciency (SCID), phenylketonuria (PKU), α-1 antitrypsin deficiency, Duchenne amyotrophy, ornithine transcarbamylase deficiency or osteogenesis imperfecta by the function of replacing or replenishing the dcc gene that causes disease.Perhaps, its can encode optionally express cell toxic agent or prodrug saccharase in for example pernicious cancer cells of target cell is to kill described cell.This application and many other application are well known to a person skilled in the art, and the described the present invention of those skilled in the art know that strengthens the cognation that therapeutic nucleic acids is expressed.
Most preferably described carrier comprises SEQ ID Nos 1 or 2, CMV promotor, multiple clone site, any one in the gene of the selected marker thing of the controlling elements control that polyadenylic acid sequence and coding are fit to.
Also provide transfection that the host cell of carrier is arranged.Eukaryotic cell preferably, more preferably mammalian cell, optimum is chosen or rodent cells.
Perhaps, may be vegetable cell.
Another aspect of the present invention is that any isolating polynucleotide, CpG island ( SEQ ID Nos 1 or 2 is disclosed especially), the disclosed carrier of above-mentioned treatment or the host cell that does not have the extension of methylating are in the especially application in gene therapy of treatment.
The present invention also provides polynucleotide, carrier or host cell to be used for the application of gene therapy medicine or composition in preparation.
The present invention also provides methods of treatment, comprises that the patient to this treatment of needs uses polynucleotide described of the present invention, carrier or the host cell of effective dose.Preferred patient suffers from can be by the disease of gene therapy.
The present invention also provides pharmaceutical composition, and it comprises polynucleotide and/or carrier and/or host cell, randomly is used for the treatment of disease with pharmaceutical carrier or mixing diluents or the cell of the particular organization with useful albumen or function is provided.
Polynucleotide of the present invention, carrier or host cell or pharmaceutical composition can comprise the interior and partial direct injection of (solid or liquid), part, eye, rectum, intraperitoneal and/or sheath of whole body intramuscular, intravenously, aerosol, mouth with following administration.
The dosage mode is incited somebody to action accurately, certainly, need be determined by concrete patient's concrete clinician, and this controls according to the albumen of required genetic expression with as the definite character of the types of organization for the treatment of target conversely.
Dosage depends on that also disease refers to disease and route of administration.The number of dosage depends on disease and from the efficacy data of clinical trial.
The amount of sending the polynucleotide that are used for the efficient gene treatment or carrier DNA according to the present invention is preferably within the scope of 50ng-1000 μ g carrier DNA/kg body weight; More preferably in the scope of about 1-100 μ g carrier DNA/kg.
Be used for the cells in vivo absorption although preferably use polynucleotide, carrier or host cell according to the present invention to Mammals, can utilize stripped method, whereby cell is taken out from animal,, heavily be implanted to described animal then with polynucleotide or carrier transduction.Liver, for example can adopt the method for exsomatizing by taking out liver cell from animal, the liver cell that external transduction liver cell and heavily implanting is transduceed in animal (as Chowdhury etc., Science 254:1802-1805,1991 descriptions are used for rabbit, or Wilson, H μ m.Gene Ther.3:179-222,1992 descriptions are used for the people) estimate.This method can also effectively be sent different cell colonys, for example red blood corpuscle, T cell, B cell and hemopoietic stem cell in the recycle system or lymphsystem.
Aspect other, the invention provides the application in cell culture system of polynucleotide carrier of the present invention or host cell to obtain required gene product.Preferably, effable nucleic acid encoding is used for the recombinant protein of expressing in the cell in vitro culture systems.
The cell culture system that is fit to is that prior art is known, and in the document that those skilled in the art will know that complete description is arranged.The invention provides the method for producing polypeptide of the present invention, comprising:
I) provide conversion/transfection that the cell of polynucleotide of the present invention is arranged;
Ii) cultivate described cell under the situation of described polypeptide helping to produce; And
Iii) from described cell or the described polypeptide of its growing environment purifying.
Perhaps, effable genes encoding non--polypeptide product, for example RNA.This RNA can level suppress the sense-rna that specific gene is expressed after translation, maybe can be enzymatic (ribozyme) or other function, for example function of ribosome-RNA(rRNA).
Also provide no methylated CpG island polynucleotide to be used to improve the application that native gene is expressed, thereby comprising described polynucleotide are inserted into can operate the position that links to each other with native gene in the genome of cell and improve the expression of gene level according to the extension of one of SEQ ID NOs:1 or 2.
In another embodiment of the invention, the non-human transgenic's on the no methylated CpG island that comprises any polynucleotide of the present invention or carrier or any extension of the present invention animal is provided, wherein said CpG is introduced by the artificially on the island.Preparation transgenic mice (Gordon etc., Proc.Natl.Acad.Sci.USA 77:7380 (1980); Harbers etc., Nature293:540 (1981); Wagner etc., Proc.Natl.Acad.Sci.USA 78:5016 (1981); With Wagner etc., Proc.Natl.Acad.Sci.USA 78:6376 (1981), sheep, pig, chicken (referring to Hammer etc., Nature 315:680 (1985)) etc. method be that the known and design of prior art is used for the present invention.
Thisly comprise polynucleotide transgenic animal of the present invention and can also be used to the required albumen of long-term production.
The present invention also provides the Mammals model of the effect of using polynucleotide of the present invention, carrier or host cell to determine gene therapy.The Mammals model comprises that its cell contains the transgenic animal of carrier of the present invention.This animal can be for testing before carrying out the human clinical trial.
The present invention also provides the application of no methylated CpG island in producing transgenic plant of polynucleotide of the present invention and extension.
Output increases or is well known by persons skilled in the art to the production of transgenic plants that the resistance of disease, insect, arid or salt improves.The present invention also provides the transgenic plant that comprise the cell that contains polynucleotide of the present invention.Some or all ofly comprise that polynucleotide cell of the present invention can come from plant.
The invention still further relates to the application of no methylated CpG island in functional genomics of polynucleotide of the present invention, carrier or extension.Functional genomics mainly relates to the evaluation of the gene of expressing specifically in specific cell type or morbid state, and the thousands of new gene order with potential drug discovery or gene therapy purpose value is provided at present.Use the subject matter of this information development new therapy to be how to determine the function of these genes.Polypeptide of the present invention can be used in the application of multiple functional genome to determine the function of gene order.Functional genome of the present invention uses and comprises, but is not restricted to:
(1) uses polynucleotide of the present invention to obtain the antisense sequences of gene order or the continuous expression in ribozyme knockdown library, thereby determine the effect of gene inactivation pair cell phenotype.
(2) use polynucleotide of the present invention to prepare the expression library of gene order, make be delivered to produce in the cell described gene order reliably, repeatably, continuous expression.The cell of the expressing said gene sequence that obtains can be used to the method for multiple Function Identification and drug discovery.For example, the neutralizing antibody of preparation gene product; The protein product of fast purifying gene itself is used for the research of structure, function or drug screening; Or be used for drug screening based on cell.
(3) use polynucleotide of the present invention to be used for the method for mouse embryo stem cell (ES) and transgenic mice.One of strong functions genome method comprises insert construct at random in the mouse ES cells gene, described construct only just allows later medicament selection in being inserted into the gene of expression, and its can easily clone out be used for the order-checking (G.Hicks etc., Nature Genetics, 16,338-334).Therefore transgenic mice with gene knockout sudden change and new sequence can easily be used to study its function.This technology is useful to 10% little musculus cdna of good representation in the mouse ES cells at present.Mixing polynucleotide of the present invention will make these methods expand to the full gene of identifying that mouse is expressed in the construct of integrating.
Detailed Description Of The Invention
The present invention only is described with reference to following accompanying drawing at present for example, wherein;
Fig. 1 demonstration shows that BamH1, HindIII, Esp31, Tth1111 and BspEI restriction site define the collection of illustrative plates of different 4,2,1.5 and the segmental HP-1/hnRNPA2 locus in 1kb CpG island.
Fig. 2 shows the nucleotide sequence of the HP-1/hnRNPA2 locus that shows BamH1, Esp31, BspE1 and Tth111I restriction site.
Fig. 3 shows the expression that can be operationally connected to the segmental EGFP reporter gene of 4kb and 1.5kb (at both direction) CpG island.The FACScan data are expressed as the fluorescence intermediate value through 71 days.
Fig. 4 shows the expression that can be operationally connected to the segmental EGFP reporter gene of 4kb and 1.5kb (at both direction) CpG island.The FACScan data are expressed as the positive cell % through 71 days.
Fig. 5 shows the expression that can be operationally connected to the segmental EGFP reporter gene of 4kb, 1.5kb and 1kb CpG island.The FACScan data are expressed as the fluorescence intermediate value through 68 days.
Fig. 6 shows the expression that can be operationally connected to the segmental EGFP reporter gene of 4kb, 1.5kb and 1kb CpG island.The FACScan data are expressed as the positive cell % through 68 days.
Fig. 7 shows the structure of two adenovirus construct Ad.CMV-Luc-SV40p (A) and Ad.1.5kb (F) UCOE-CMV-Luc-SV40p (A), and it is based on adenoviral serotype 5.The luciferase expression box is inserted in the E1 district with direction from left to right.E1 and E3 district are deleted from virus.Because the deletion of E1, virus is replication defective.CMV (cytomegalovirus) is people's cmv enhancer/promotor.SV40p (A) is the SV40 virus polyadenylic acid signal in late period from pGL3basic (Promega).
Fig. 8 has shown that 1.5kb UCOE improves genetic expression after sending by adenovirus carrier in the HeLa cell.The HeLa cell infects in the medium with Ad.CMV-Luc-SV40p (A) and 2-3 hour (normally the HeLa medium only comprises 1%FCS) of Ad.1.5kb (F) UCOE-CMV-Luc-SV40p (A) virus infection at 200 μ l with MOI50.Add the 2ml perfect medium hatching the back, and with cell inoculation in 6 orifice plates.Infecting 2 days post analysis uciferase activities.The result who shows three independent experiments.Experiment is carried out three times and is repeated.Be shown as mean value+/-standard deviation.
Fig. 9 has shown that 1.5kb UCOE effect is irrelevant with the preparation of virus.The HeLa cell uses in the infection medium of 200 μ l (normal Hela medium only comprises 1%FCS) with MOI50 that independently the virus of A d.CMV-Luc-SV40p of virus formulation (A) and Ad.1.5kb (F) UCOE-CMV-Luc-SV40p (A) infected respectively 2-3 hour from two.After hatching, add the 2ml perfect medium, then with cell inoculation in 6 orifice plates.Infecting 2 days post analysis uciferase activities.Show a typical result of experiment.Experiment is carried out three times and is repeated.
Figure 10 shows that 1.5kb UCOE improves transgene expression level and stability in the CHO-K1 aggregate of retrovirus transduction.(A) average GFP value and the time relation of the CHO-K1 aggregate of FACS sorting (36,000 cells of initial FACS sorting) behind retrovirus transduction CMV and 1.5UCOE-CMV construct.Referred in first day to measure first day (after the FACS sorting 8 days).(B) histogram of each Measuring Time point of CHO-K1 aggregate of FACS sorting.
Figure 11 is presented at low MOI and infects the GFP expression of the CHO-K1 (aggregate I) of transduction afterwards.(A) the GFP positive (M1) CHO-K1 group is in the average GFP value of each time point of retrovirus transduction back.CMV group from the 3.19%GFP positive cell begin and 1.5UCOE-CMV group from the 4.99%GFP positive cell.(B) cell mass is at the histogram of each time point.
Figure 12 is presented at the GFP expression that medium MOI infects the CHO-K11 (aggregate II) of back transduction.(A) the GFP positive (M1) CHO-K1 group is in the average GFP value of each time point of retrovirus transduction back.CMV group from the 25.2%GFP positive cell begin and 1.5UCOE-CMV group from the 14.35%GFP positive cell.(B) cell mass is at the histogram of each time point.
Figure 13 shows that 1.5kb UCOE improves transgene expression level and stability in the HeLa aggregate of retrovirus transduction.(A) average GFP value and the time relation of the HeLa aggregate of FACS sorting (10,000 cells of initial FACS sorting) behind retrovirus transduction CMV and 1.5UCOE-CMV construct.Referred in first day to measure first day (after the FACS sorting 5 days).(B) the HeLa aggregate of FACS sorting is at the histogram of each Measuring Time point.
Figure 14 shows that 1.5kbUCOE improves the consistence that GFP expresses between the HeLa cell clone neutralization of retrovirus transduction.The HeLa of FACS sorting clone is extended to be 6 hole sizes and to analyze GFP and express.(A-C) all CMV clones' histogram.(D-E) all 1.5UCOE-CMV clones' histogram.
Figure 15 shows that 1.5kb UCOE reduces the variation coefficient (CV) that GFP expresses in the HeLa cell clone.The average CV value of whole retrovirus HeLa clone's transduction and the FACS sorting of demonstration Fig. 5 mean value (+/-SD).1.5UCOE-CMV comparing significantly with the CMV clone, clone's CV and its standard deviation (SD) reduce.
Embodiment
Embodiment 1A 1.5kb HP-1/hnRNP A2UCOE strengthens expression
Material and method
The structure of carrier
As we described in the first to file WO 00/05393, (Stratagene) produces support C ET20 among the pBluescript by the HindIII fragment (it comprises the CpG island of HP1/RNP promotor and extension) of the 8.3kb of people HP-1/hnRNP A2 locus is cloned into.
4186bp (the being called 4kb) fragment of inserting is removed by BamH1 and HindIII digestion then.It is flat terminal that these fragments use the T4DNA polysaccharase to mend, and be connected to (Clontech) among the pEGFPN-1 of Asel digestion and reuse the T4 archaeal dna polymerase and mend flat terminal.Separate segmental clone then with both direction.
The Esp31 of 1546bp (isoschizomers of BsmBI) fragment (being called the 1.5kb fragment) is separated from CET20 by Esp31 (BsmBI) digestion once more, mend flat terminal subsequently, then these are connected to the Asel site of aforesaid pEGFPN-1, identify the segmental clone of both direction (" just " to the RNPA2 promotor have with transcribe the genetically modified adjacent downstream operation of EGFP CMV promotor identical 5 ' to 3 ' direction.)
Transfection
According to standard method transfection CHO-K1 cell by in G418, being selected, as the description of the pending application of introducing reference herein.
The GFP expression analysis
Transfectional cell is cultivated at 600 μ g/ml G418 and is selected in the substratum.Cell is peeled off with trypsinase/EDTA and matrix with the standard phosphate buffered saline washing and according to standard method.Add excessive nutritional blend F12 (HAM) substratum (Gibco), and change cell over to 5ml round bottom polystyrene test tube and be used for Becton Dickinson FACScan and analyze.Detect GFP fluorescence and with parental cell group's autofluorescence relatively.Expression in the cell colony both can be expressed as with respect to contrasting (according to standard method) fluorescence intermediate value of the arbitrary unit group of linear scale in the drawings, also can be expressed as the % of the cell that is accredited as positive expression.
The result
As shown in Figure 3,, when 1.5kb fragment forward inserts, express significantly enhancing (about 10 times continue at least 70 days) compared with the control, observe enhancing about 60% during the 4kb fragment from fluorescence intermediate value angle.In the experiment that Fig. 5 shows, can be suitable by the segmental expression of forward 1.5kb with the expression that obtains with the 4kb fragment.Yet reverse direction shows as from fluorescence intermediate value angle less effectively, as shown in Figure 3.
Fig. 4 shows that both direction is suitable from positive cell % angle.Yet, these data presentation fragments perhaps have to a certain degree directivity and, for most applications, forward is preferred.
Embodiment 2A 1kb HP-1/hnRNP A2 UCOE strengthens expression
Material and method
The structure of carrier
The carrier that contains 1kb UCOE is removed 5 ' 500bp by having the segmental pEGFPN-1 carrier of 1.5kbEsp31 with PciI and BspEI digestion forward, to mend gentle reconnecting.This only produces has the segmental carrier of 987bp BspEl-Esp31 in a direction.
The result
Described forward 1kb (987bp) fragment shows as can be suitable with segmental fluorescence intermediate value of forward 1.5kb (Fig. 5) and positive cell % (Fig. 6)
In the construct of embodiment 3A 1.5kb HP-1/hnRNP A2 UCOE enhancing adenovirus coding
Express
Material and method
Cell cultures
HeLa is available from ATCC (Manassas, Virginia).PER.C6 is available from Crucell, (Leiden, Holland).The method that the clone of portfolio acquisition such as manufacturer are recommended is cultivated.911 cells are so kind as to give by L.S.Young professor (Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham, Britain), and cultivate in containing antibiotic DMEM/10%FCS.
Plasmid construction
PGL3basic comprises luciferase-SV40p (A) box available from Promega (Madison, WI, the U.S.) and in the multiple clone site downstream.People's cmv enhancer/promotor (0.9kb) is cloned among the pGL3basic of Smal digestion and is prepared pGL3/CMV-Luc-SV40p (A).Be preparation pGL3/1.5kb (F) UCOE-CMV-SV40p (A), 1.5kb UCOE Esp3I fragment (referring to Fig. 1 and 2) is put down endization (NEB with the T4DNA polysaccharase, Beverly, MA USA) is connected to putting down among the pGL3basic/CMV-Luc of endization with T4 of NheI digestion then.
Virus vector makes up
For making up pPS1128/CMV-Luc-SV40p (A) and pPS1128/1.5kb (F) UCOE-CMV-Luc-SV40p (A), expression cassette excises from pGL3/CMV-Luc-SV40p (A) by the digestion of PvuI/NheI/BamHI and excises from pGL3/1.5kb (F) UCOE-CMV-Luc-SV40p (A) by the digestion of KpnI/BamHI, two expression cassettes are then by flat endization be cloned among the pPS1128 of the peaceful endization that SpeI digests (Djeha etc., Cancer Gene Therapy 2000:721-731).Plasmid is analyzed by Restriction Enzyme digestion.
Virus of A d.CMV-Luc-SV40p (A) and Ad.1.5kb (F) UCOE-CMV-Luc-SV40p (A) make up (Djeha etc. by use pPS1128/CMV-Luc-SV40p (A) and pPS1128/1.5kb (F) UCOE-CMV-Luc-SV40p (A) and overlapping adenovirus skeleton carrier pPS1160 homologous recombination respectively in PER.C6, gene therapy for cancer 2000:721-731), description (the Lipinski etc. in amplification and CsCI purifying and titer determination such as other places, Gene Therapy, 8,2001:274-281).
Virus infection and uciferase activity analysis
HeLa cell (per 6 holes 5.0 * 10
4) infect medium suspension (containing antibiotic EMEM/1%FCS) infection 2-3 hour with the 200 μ l that contain corresponding virus.Add the 1ml perfect medium then, in cell inoculation to 6 orifice plate.By at 200 μ l lysis buffer (10mM sodium phosphate pH 7.8,8mM MgCl
2, 1mM EDTA, 1%Triton-X-100,15% glycerine) in lysing cell prepare full cell extract and (1 minute, 13,000 * g RT) obtained clarifying lysate by centrifugal.Use photometer (Lumat LB 9501, Berthold, Wildbad, Germany) to analyze the uciferase activity of each portion supernatant liquor at indicated time point in linearity range.
The result
Whether can in adenovirus carrier, improve genetic expression for analyzing UCOE, 1.5kbUCOE is cloned into the people's cmv enhancer/promotor upstream (the RNP promotor has the direction identical with the CMV promotor) that drives luciferase reporter gene with forward.Fig. 7 schematically shows the structure of the virus of two their uciferase activities of comparison.The HeLa cell is with the corresponding virus infection of MOI 50, and the uciferase activity after 2 days is infected in analysis.Fig. 8 is clear, and the 1.5kbUCOE fragment that shows can greatly increase the expression level of reporter gene in the HeLa cell.For getting rid of the specific effect relevant with virus formulation, we prepare the new preparation of two viruses and repeat this experiment.Fig. 9 has shown that the UCOE effect does not depend on virus formulation and utilizes new independent preparation can repeat fully.
Embodiment 4A 1.5kb HP-1/hnRNP A2UCOE strengthens the construct of retrovirus coding
In expression
Material and method
Cell cultures
HeLa, CHO-K1 and 293 is available from ATCC (Manassas, Virginia).HeLa and 293 two cultivate and are adding 1%NEAA and containing among the DMEM of antibiotic 10%FCS.CHO-K1 cultivates in containing mixing F12 (HAM) nutritional medium of 10%FCS and antibiotic nutrition.
Plasmid construction
Plasmid pVPack-GP and pVPack-VSV-G are available from Stratagene (LaJolla, CA, the U.S.) and express retrovirus gag-pol and tunicle VSV-G albumen respectively.Retroviral vector pQCXIX is available from BD Bioscience, and it comprises the people CMV promotor (Palo Alto, CA, the U.S.) that is used for transgene expression.Plasmid phrGFP-1 is available from Stratagene and be used as the source of the GFP cDNA of modification.Be clone pQCXIX-CMV-hrGFP, hrGFP cDNA is excised and is cloned into the pQCXIX of BamHI/EcoRV digestion by BamHI/EcoRV digestion from phr-GFP-1.Be to produce pQCXIX-1.5UCOE-CMV-hrGFP, with 1.5kbUCOE fragment (Esp3I/BsmBI fragment) as flat endization fragment cloning to also putting down among the pQCXIX-CMV-hrGFP of endization that XbaI digests.
The preparation of two preferendum VSV-G-tunicle retrovirus particles
All virus is made pseudovirus with two preferendum VSV-G (vesicular stomatitis virus glycoprotein) that can produce efficient and wide host range transduction.The retroviral vector that uses produces the virus of self-passivation, and this is owing to therefore 3 ' LTR U3 district the deletion that copies 5 ' LTR after reverse transcription to suppresses further to transcribe from any of 5 ' LTR.
4.5 * 10
6293 cells (second day cell should 80-90% be paved with) are seeded in the transient transfection day before yesterday of 8.4 cm diameter (55.6cm with type i collagen enzyme bag quilt
2) in the plate.μ l Lipofectamine 2000 (Invitrogen, Groenningen, Holland) mixed to be incorporated under the room temperature (RT) with 286 μ l OptiMEM (Invitrogen) and was incubated 5 minutes next day 114.Abreast, each plasmid pVPack-GP, pVPack-VSV-G and the retroviral vector with 1.5 μ g mixes with OptiMEM to reach final volume 400 μ l.The DNA mixture adds in Lipofectamine 2000 solution and RT insulation 20-30 minute.293 cells wash once and add to cell the OptiMEM/10%FCS of 6ml with PBS between incubation period.Last DNA/Lipofectamine mixture is added dropwise to cell, and cell is at 5%CO
2Cultivated 5 hours for 37 ℃ in the incubator.Substitute substratum with 293 fresh substratum of 8ml then, cell was continued to cultivate 24-36 hour.At last, contain the supernatant liquor of virus particle from cell harvesting, with sedimentation cell and cell debris, supernatant liquor used the low protein bound 0.45 μ m filter filtration sterilization (Millipore, Molsheim, France) of Millex-HV PVDF centrifugal 5 minutes of 1000 RPM.Supernatant liquor is divided into aliquot, and liquid nitrogen is freezing rapidly and be stored in-80 ℃.
Retrovirus transduction target cell system
(HeLa CHO-K1) is infecting the day before yesterday by with l * 10 to target cell
5Cells/well is inoculated in 6 orifice plates.Be the virus transduction, have 1 of 8.0 μ g/ml, 5-dimethyl l, 5-phenodiazine 11 methylene radical gather the supernatant liquor that contains virus that adds different amounts under the Methobromide (Sigma, St.Louis, the Missouri State, the U.S.).Cell and virus culture 24 hours are replaced substratum with fresh substratum then.Genetic expression can be observed in transduction in back two days.For preventing the multiple copied effect of every cell, during analyzing, all use the virus quantity target cell infection that produces far fewer than 100% transduction efficiency (giving usually and the 1-20%GFP positive cell).In this transduction efficiency scope, the number linear dependence of the volume of the supernatant liquor that is used to infect and positive cell and average expression level shows much lower increase.
Facs analysis
For carrying out facs analysis, target cell is washed with PBS, carry out trypsin acting and be resuspended in the perfect medium, with Becton Dickinson FACScan (BD, Franklin Lakes, NJ, U.S.) being used for each constant instrument set(ting)value of analyzing analyzes hrGFP (green fluorescent protein) and expresses.CeIIQuest software analysis data with Apple Mcintosh.
The FACS sorting
Be that sorting expresses HeLa and the CHO-K1 cell of GFP, cell is produced and is used for facs analysis and uses BD Bioscience FACS sorter sorting (being so kind as to give University of Birmingham, Britain by The Institute for CancerStudies) then.The one clone and the aggregate of sorting are amplified subsequently, experience for some time subsequently and express GFP.
The result
In first research, the CHO-K1 cell is transduceed with the supernatant liquor that contains retrovirus CMV-hrGFP (CMV) and 1.5UCOE CMV-hrGFP (1.5UCOE-CMV) cell.The description of CHO-K1 cell such as material and method is transduceed, and infects after three days the GFP positive cell and is divided by FACS and elect the zoarium of trooping (each cell construction body 36,000 cell) as and increase then.Carry out through for some time that GFP expresses and the histogram mapping also is illustrated among Figure 10 A and the B respectively.At the 17th day that analyzes, the mean value of 1.5UCOE-CMV aggregate was higher 2.6 times than the mean value of CMV aggregate.In addition, shown in the histogram of Figure 10 B, the 1.5UCOE-CMV aggregate produces more intensive GFP than CMV aggregate and expresses the peak.The average CV value (variation coefficient of GFP positive cell; This is the mark that GFP expresses homogeneity) in Marker 1, be 146.0 and be 83.0 to the 1.5UCOE-CMV aggregate to the CMV aggregate.
Research is compared therewith, and transduction CHO-K1 is through for some time research cell mass but do not carry out the FACS sorting.Select to produce the MOI of low per-cent positive cell to get rid of the multiple copied effect of every cell.Shown in Figure 11 A (the aggregate I that about 5%GFP positive cell is arranged), the 1.5UCOE-CMV group in the end average of the positive cell of Measuring Time point is higher 2.8 times than CMV group's average.For aggregate II (about 15-25%GFP positive cell; Figure 12 A), 1.5UCOE-CMV group is higher 2.3 times than CMV group's average at the mean value of the positive cell of Measuring Time point at the latest.
With the faciation ratio of FACS sorting, 1.5UCOE-CMV group produces obviously more intensive expression peak (Figure 11 B and Figure 12 B) with the CMV faciation than putting in all analysis times.
As for CHO-K1, transduction stably has the aggregate of 10,000 HeLa cells of retroviral construct body to carry out the FACS sorting.At Figure 13 A, average GFP value is shown as the mean value that comprises the FACS sorting same day (the 5th day).In measuring the last time, the mean value of 1.5UCOE-CMV aggregate is higher 1.6 times than the mean value of CMV aggregate.Shown in the histogram of Figure 13 B, many CMV cells lose its high GFP expression level after for some time; Opposite most of 1.5UCOE-CMV cell keeps its high GFP to express.
The HeLa single cell clone carry out equally that FACS-selects and 6 orifice plates that increase in.The CMV clone trends towards producing the high slightly expression peak than the 1.5UCOE-CMV clone, yet, no matter inner still between the clone the clone, shown in the clone's of whole analyses histogram (Figure 14 A-E), owing to there is UCOE, the consistence of expression level is increased significantly.Figure 15 shows mean coefficient (CV) and its standard difference (SD) of GFP differential expression.CMV clone's CV+/-the SD value is 135.0+/-165.3, and the 1.5UCOE-CMV clone is 49.4+/-10.9.
Sequence table
＜110〉Ml Lab PLC (ML Laboratories PLC)
＜120〉improved Expression element (Improved expression ements)
<130>p101405
<160>2
<170>PatentIn?version?3.1
<210>1
<211>1546
<212>DNA
<213>Homo?sapiens
ggccctccgc?gcctacagct?caagccacat?ccgaaggggg?agggagccgg?gagctgcgcg 60
cggggccgcc?ggggggaggg?gtggcaccgc?ccacgccggg?cggccacgaa?gggcggggca 120
gcgggcgcgc?gcgcggcggg?gggaggggcc?ggcgccgcgc?ccgctgggaa?ttggggccct 180
agggggaggg?cggaggcgcc?gacgaccgcg?gcacttaccg?ttcgcggcgt?ggcgcccggt 240
ggtccccaag?gggagggaag?ggggaggcgg?ggcgaggaca?gtgaccggag?tctcctcagc 300
ggtggctttt?ctgcttggca?gcctcagcgg?ctggcgccaa?aaccggactc?cgcccacttc 360
ctcgcccgcc?ggtgcgaggg?tgtggaatcc?tccagacgct?gggggagggg?gagttgggag 420
cttaaaaact?agtacccctt?tgggaccact?ttcagcagcg?aactctcctg?tacaccaggg 480
gtcagttcca?cagacgcggg?ccaggggtgg?gtcattgcgg?cgtgaacaat?aatttgacta 540
gaagttgatt?cgggtgtttc?cggaaggggc?cgagtcaatc?cgccgagttg?gggcacggaa 600
aacaaaaagg?gaaggctact?aagatttttc?tggcgggggt?tatcattggc?gtaactgcag 660
ggaccacctc?ccgggttgag?ggggctggat?ctccaggctg?cggattaagc?ccctcccgtc 720
ggcgttaatt?tcaaactgcg?cgacgtttct?cacctgcctt?cgccaaggca?ggggccggga 780
ccctattcca?agaggtagta?actagcagga?ctctagcctt?ccgcaattca?ttgagcgcat 840
ttacggaagt?aacgtcgggt?actgtctctg?gccgcaaggg?tgggaggagt?acgcatttgg 900
cgtaaggtgg?ggcgtagagc?cttcccgcca?ttggcggcgg?atagggcgtt?tacgcgacgg 960
cctgacgtag?cggaagacgc?gttagtgggg?gggaaggttc?tagaaaagcg?gcggcagcgg 1020
ctctagcggc?agtagcagca?gcgccgggtc?ccgtgcggag?gtgctcctcg?cagagttgtt 1080
tctcgagcag?cggcagttct?cactacagcg?ccaggacgag?tccggttcgt?gttcgtccgc 1140
ggagatctct?ctcatctcgc?tcggctgcgg?gaaatcgggc?tgaagcgact?gagtccgcga 1200
tggaggtaac?gggtttgaaa?tcaatgagtt?attgaaaagg?gcatggcgag?gccgttggcg 1260
cctcagtgga?agtcggccag?ccgcctccgt?gggagagagg?caggaaatcg?gaccaattca 1320
gtagcagtgg?ggcttaaggt?ttatgaacgg?ggtcttgagc?ggaggcctga?gcgtacaaac 1380
agcttcccca?ccctcagcct?cccggcgcca?tttcccttca?ctgggggtgg?gggatgggga 1440
gctttcacat?ggcggacgct?gccccgctgg?ggtgaaagtg?gggcgcggag?gcgggaattc 1500
ttattccctt?tctaaagcac?gctgcttcgg?gggccacggc?gtctcc 1546
<210>2
<211>987
<212>DNA
<213>Homo?sapiens
<400>2
ccggaagggg?ccgagtcaat?ccgccgagtt?ggggcacgga?aaacaaaaag?ggaaggctac 60
taagattttt?ctggcggggg?ttatcattgg?cgtaactgca?gggaccacct?cccgggttga 120
gggggctgga?tctccaggct?gcggattaag?cccctcccgt?cggcgttaat?ttcaaactgc 180
gcgacgtttc?tcacctgcct?tcgccaaggc?aggggccggg?accctattcc?aagaggtagt 240
aactagcagg?actctagcct?tccgcaattc?attgagcgca?tttacggaag?taacgtcggg 300
tactgtctct?ggccgcaagg?gtgggaggag?tacgcatttg?gcgtaaggtg?gggcgtagag 360
ccttcccgcc?attggcggcg?gatagggcgt?ttacgcgacg?gcctgacgta?gcggaagacg 420
cgttagtggg?ggggaaggtt?ctagaaaagc?ggcggcagcg?gctctagcgg?cagtagcagc 480
agcgccgggt?cccgtgcgga?ggtgctcctc?gcagagttgt?ttctcgagca?gcggcagttc 540
tcactacagc?gccaggacga?gtccggttcg?tgttcgtccg?cggagatctc?tctcatctcg 600
ctcggctgcg?ggaaatcggg?ctgaagcgac?tgagtccgcg?atggaggtaa?cgggtttgaa 660
atcaatgagt?tattgaaaag?ggcatggcga?ggccgttggc?gcctcagtgg?aagtcggcca 720
gccgcctccg?tgggagagag?gcaggaaatc?ggaccaattc?agtagcagtg?gggcttaagg 780
tttatgaacg?gggtcttgag?cggaggcctg?agcgtacaaa?cagcttcccc?accctcagcc 840
tcccggcgcc?atttcccttc?actgggggtg?ggggatgggg?agctttcaca?tggcggacgc 900
tgccccgctg?gggtgaaagt?ggggcgcgga?ggcgggaatt?cttattccct?ttctaaagca 960
cgctgcttcg?ggggccacgg?cgtctcc 987
Claims (26)
1. isolating polynucleotide comprise
A) the no methylated CpG island of Yan Shening;
B) effable open reading frame can be operationally connected to the no methylated CpG island of described extension;
C) promotor can be operationally connected to described open reading frame, and wherein said promotor is not connected to described CpG island natively;
It is characterized in that described CpG island is no more than repeating to be expressed in the two or more types of organizations and obtaining of 2kb size and wherein said open reading frame.
2. the polynucleotide of claim 1, wherein said CpG island comprises the fragment in HP-1/hnRNPA2 site.
3. the polynucleotide of claim 2, wherein said fragment is a people HP-1/hnRNPA2 locus.
4. each polynucleotide of claim 1 to 3 comprise the fragment of the HP-1/hnRNPA2 locus that is no more than 1.6kb.
5. the polynucleotide of claim 4 comprise the Esp31 restriction fragment.
6. the polynucleotide of claim 5 comprise the sequence of Fig. 2, Nucleotide 977 to 2522 (SEQ ID NO:1) or its functional analogue.
7. each polynucleotide of claim 1 to 6 comprise the fragment of the HP-1/hnRNPA2 locus that is no more than 1kb.
8. the polynucleotide of claim 7 comprise the BspE1-Esp31 restriction fragment.
9. the polynucleotide of claim 8 comprise the sequence of Fig. 2, Nucleotide 1536 to 2522 (SEQ ID NO:2) or its functional analogue.
10. carrier contains right and requires 1 to 9 each polynucleotide.
11. the carrier of claim 10, carrier wherein is an episomal vector.
12. the carrier of claim 10, carrier wherein is an integrating vector.
13. each carrier of claim 10-12, carrier wherein is a plasmid.
14. each carrier of claim 10 to 13, wherein the gene that can be operatively connected is a therapeutic nucleic acids.
15. each carrier of claim 10 to 14 comprises following any: SEQ IDNOs 1 or 2, CMV promotor, multiple clone site, polyadenylic acid sequence and the gene of coding selected marker thing under suitable controlling elements.
16. host cell comprises each polynucleotide of claim 1 to 9, or each carrier of claim 10 to 15.
17. claim 1 to 9 each isolating polynucleotide, claim 10 to 15 each carrier or the host cell of claim 16, be used for gene therapy.
18. each each carrier or the application that is used for gene therapy medicine in preparation of the host cell of claim 16 of polynucleotide, claim 10 to 15 of claim 1 to 9.
17. methods of treatment, comprise patient to this treatment of needs use claim 1 to 9 each effective dose polynucleotide, claim 10 to 15 each carrier or the host cell of claim 16.
18. pharmaceutical composition, with the claim 1 to 9 of pharmaceutical excipient combination each polynucleotide, claim 10 to 15 each carrier or the host cell of claim 16.
19. claim 1 to 9 each polynucleotide, claim 10 to 15 each carrier or the host cell of claim 16, in cell culture system for obtaining the application of required gene product.
20.SEQ the application of the polynucleotide of ID NOs 1 or 2 in improving endogenous gene expression is included in the position that can be operatively connected endogenous gene polynucleotide is inserted in the genome of cell, thereby improves the expression of gene level.
21. transgenic nonhuman animal comprises and contains each polynucleotide or each the cell of polynucleotide of carrier of claim 10 to 15 of claim 1 to 9 that wherein said polynucleotide are manually introduced.
22. claim 1 to 9 each polynucleotide, claim 10 to 15 each carrier or the host cell of claim 16 in the expression that obtains the inverted defined gene sequence with the application in the expression of the gene order of inactivation correspondence.
23. each each the application of polynucleotide in the preparation expression library of carrier of polynucleotide, claim 10 to 15 of claim 1 to 9.
24.SEQ the application of the polynucleotide of ID NOs 1 or 2 in the method for identifying the expressible gene in the non-human animal, comprise that the construct that will contain polynucleotide is inserted in non-human animal's the embryonic stem cell, wherein said construct can allow to carry out medicament selection after inserting the gene of expressing.
Applications Claiming Priority (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0302330.6 | 2003-02-01 | ||
| GB0302330A GB0302330D0 (en) | 2003-02-01 | 2003-02-01 | Improved expression elements |
| GB0308503A GB0308503D0 (en) | 2003-04-12 | 2003-04-12 | Improved expression elements |
| GB0308503.2 | 2003-04-12 | ||
| GB0320824A GB0320824D0 (en) | 2003-09-05 | 2003-09-05 | Improved expression elements |
| GB0320824.6 | 2003-09-05 | ||
| PCT/GB2004/000387 WO2004067701A2 (en) | 2003-02-01 | 2004-02-02 | Improved genetic elements providing high levels of expression |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1748034A true CN1748034A (en) | 2006-03-15 |
| CN1748034B CN1748034B (en) | 2011-03-02 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200480003321.1A Expired - Lifetime CN1748034B (en) | 2003-02-01 | 2004-02-02 | Improved expression element |
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| CN (1) | CN1748034B (en) |
| GB (1) | GB0302330D0 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100368549C (en) * | 2006-03-29 | 2008-02-13 | 北京未名凯拓作物设计中心有限公司 | Bacterial plasmid, its derivative plasmid and application |
| CN109415730A (en) * | 2016-06-13 | 2019-03-01 | 北卡罗来纳大学教堂山分校 | The CLN1 gene and expression cassette of optimization and their application |
| CN112575031A (en) * | 2019-09-29 | 2021-03-30 | 新乡医学院 | Ubiquitous chromatin open expression element, recombinant expression vector, expression system, preparation method and application thereof |
-
2003
- 2003-02-01 GB GB0302330A patent/GB0302330D0/en not_active Ceased
-
2004
- 2004-02-02 CN CN200480003321.1A patent/CN1748034B/en not_active Expired - Lifetime
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100368549C (en) * | 2006-03-29 | 2008-02-13 | 北京未名凯拓作物设计中心有限公司 | Bacterial plasmid, its derivative plasmid and application |
| CN109415730A (en) * | 2016-06-13 | 2019-03-01 | 北卡罗来纳大学教堂山分校 | The CLN1 gene and expression cassette of optimization and their application |
| CN112575031A (en) * | 2019-09-29 | 2021-03-30 | 新乡医学院 | Ubiquitous chromatin open expression element, recombinant expression vector, expression system, preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1748034B (en) | 2011-03-02 |
| GB0302330D0 (en) | 2003-03-05 |
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