WO2013006142A1 - Nouveau procédé et réactif autorisant des modifications génétiques rapides dans des cellules eucaryotes - Google Patents

Nouveau procédé et réactif autorisant des modifications génétiques rapides dans des cellules eucaryotes Download PDF

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WO2013006142A1
WO2013006142A1 PCT/SG2012/000238 SG2012000238W WO2013006142A1 WO 2013006142 A1 WO2013006142 A1 WO 2013006142A1 SG 2012000238 W SG2012000238 W SG 2012000238W WO 2013006142 A1 WO2013006142 A1 WO 2013006142A1
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nucleic acid
seq
site
cells
shrna
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PCT/SG2012/000238
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Eugene Makeyev
Piyush KHANDELIA
Karen YAP
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Nanyang Technological University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome

Definitions

  • the present invention relates to the field of molecular and cell biology, more specifically genetic alterations in eukaryotic cells.
  • RNA interference sequence-specific gene silencing by double-stranded (ds) RNA molecules
  • ds double-stranded
  • Two altemitive molecules are routinely used to trigger RNAi in mammalian cells: (i) chemically or enzymatically synthesized/generated small interfering (si) RNAs and (ii) genetically encoded short hairpin (sh) RNAs that are converted into siRNAs by the cellular RNAi machinery (2-6).
  • shRNAi RNA interference
  • the relatively low cost and the possibility of inducing sustained and tunable silencing, combined with the minimized risk of off-target effects make the shRNA approach especially attractive for high through put loss-of-function screens and developing RNAi-based therapies (6-9).
  • obtaining populations of homogeneous shRNA-expressing cell normally entails time-consuming and labor intensive enrichment or cell cloning steps.
  • the recently published lenti viral toolkits for inducible shRNA expression generate populations containing substantial fractions of cells expressing shRNAs at low levels, which requires additional fluorescence-activated cell sorting (FACS) steps to improve the overall knockdown efficiency (10, 15).
  • FACS fluorescence-activated cell sorting
  • shRNA-encoding virus vectors require considerable efforts to prepare high-titre stocks and importantly, are associated with biosafety concerns and a substantial bureaucratic burden.
  • RMCE Recombination-mediated cassette exchange
  • transgenes directionally into predefined genome sites.
  • a common feature of the system is that each consists of a single polypeptide recombinase Cre, FLP or R and two identical or almost identical palendromic recognition sites lox, FRT or RS. For example using two identical but oppositely orientated RS sites.
  • RMCE can use both the Cre//ox and FLPIFRT systems in animal cell cultures. [0010].
  • RMCE has recently been used for stable shRNA expression in HeLa cells (17-19). However, a relatively high incidence of unspecific integration events still called for additional cloning steps-to isolate shRNA-expressing cells from the original recombinant pools (17-19). DNA excision can subsequently happen between any pair of compatible sites and result in the restoration of the original two DNA molecules or the exchange of the intervening DNA segments between the two DNA molecules.
  • the number of acceptor cell lines suitable for RMCE is rather limited.
  • the present invention seeks to ameliorate at least one of the problems mentioned above.
  • a first aspect of the invention includes a process for obtaining a nearly homogenous population of genetically altered eukaryotic cells, said process comprising:
  • first selectable marker protein-coding sequence comprising a first selectable marker protein-coding sequence, wherein the isolated nucleic acid fragment is flanked by a first recombination site and a second non-identical recombination site;
  • cassette comprises a strong polyadenylation site operably linked to a second isolated nucleic acid fragment comprising a targeting nucleic acid site and a second selectable marker protein-coding sequence wherein the second isolated nucleic acid fragment is flanked by the first recombination site and the second non-identical recombination site; c. providing a recombinase that recognizes and implements
  • the strong polyadenylation site is a modified polyadenylation signal from thymidine kinase gene SEQ ID NO: 52.
  • the targeting nucleic acid site comprises an shRNA directed to a 3' untranslated region of a target nucleic acid of interest.
  • Some embodiments include an shRNA sequence selected from any one of SEQ ID NO: 1 , SEQ ID NO: 2 and SEQ ID numbers 54 to SEQ ID NO: 175 Listed in table 2.
  • the non-identical recombination sites are two similar or almost identical palendromic recognition sites such as lox, FRT or RS.
  • the non-identical recombination sites are Lox2272 (SEQ ID NO: 48), and LoxP (SEQ ID NO: 49).
  • the recombinase comprises transiently expressing within the cell an expression plasmid comprising a polynucleotide encoding said
  • any recombinase enzyme known in the art to catalyse a site specific recombination event between two nucleic acid recognition sites would be suitable provided the recombinase is able to recognize the two nucleic acid recognition sites.
  • Examples include a Cre recombinase with Lox recognition sites or a Flippase recombination enzyme (FLP) with Frt recognition sites.
  • the said recombinase is a Cre amino acid.
  • the Cre has been modified to include an N-terminal nuclear localization signal (SEQ ID NO: 50). This may enhance the efficiency of the recombination.
  • the transgenic process of the invention ensures that transformed cells express a selectable marker.
  • Cells that lack a functional selectable marker gene will be killed by the selection agent.
  • Selectable marker genes include genes conferring resistance to antibiotics, herbicidal compounds.
  • a specific selection agent may have one or more corresponding selectable marker genes.
  • a specific selectable marker gene may have one or more corresponding selection agents.
  • the first selectable marker protein-coding sequence encodes a blasticidin-resistant protein that has an amino acid sequence comprising SEQ ID NO: 51.
  • the second selectable marker protein-coding sequence encodes a puromycin-resistance protein that has an amino acid sequence comprising SEQ ID NO: 53 wherein the selection agent is puromycin.
  • the first isolated nucleic acid fragment further comprises at least one strong constitutive promoter operably linked to the targeting nucleic acid site.
  • the at least one strong constitutive promoter operably linked to the targeting nucleic acid site is EF1 a (SEQ ID No. 47).
  • Another aspect of the invention includes a nearly homogenous population of genetically altered eukaryotic cells, having stably incorporated in its genome a donor cassette comprises a strong polyadenylation site operably linked to a isolated nucleic acid fragment comprising a targeting nucleic acid site and a selectable marker protein-coding sequence wherein the isolated nucleic acid fragment is flanked by a first recombination site and a second non-identical recombination site.
  • the targeting nucleic acid site comprises an shRNA directed to a 3' untranslated region of a target nucleic acid of interest.
  • the shRNA may be selected from any one of SEQ ID NO: 1 , SEQ ID NO: 2 and SEQ ID numbers 54 to 175 listed in table 2.
  • non-identical recombination sites are as described above.
  • the strong polyadenylation site is as described above.
  • selectable marker protein-coding sequence is as described above.
  • Another aspect of the invention includes a kit for obtaining a nearly homogenous population of genetically altered eukaryotic cells, comprising:
  • a vector comprising a first isolated nucleic acid fragment comprising a first selectable marker protein-coding sequence, wherein the isolated nucleic acid fragment is flanked by a first recombination site and a second non-identical recombination site;
  • polyadenylation site operably linked to a second isolated nucleic acid fragment comprising a targeting nucleic acid site and a second selectable marker protein-coding sequence wherein the second isolated nucleic acid fragment is flanked by the first recombination site and the second non-identical recombination site;
  • a plasmid comprising a recombinase that recognizes the non-identical recombination sites
  • the targeting nucleic acid site of the kit comprises an shRNA directed to a 3' untranslated region of a target nucleic acid of interest.
  • the shRNA may be selected from any one of SEQ ID NO: 1 , SEQ ID NO: 2 and SEQ ID numbers 54 to 75 listed in table 2.
  • the non-identical recombination sites are as described above.
  • the strong polyadenylation site of the kit is as described above.
  • the selectable marker protein-coding sequence and the selection agent of the kit are as described above.
  • the recombinase of the kit is as described above.
  • the first isolated nucleic acid fragment of the kit further comprises at least one strong constitutive promoter operably linked to the targeting nucleic acid site.
  • the at least one strong constitutive promoter operably linked to the targeting nucleic acid site is EF1a (SEQ ID No. 47)
  • Figure 1 Flowchart of a typical HILO-RMCE experiment.
  • Figure 2 Diagram of the HILO-RMCE reaction using the pRD1 donor plasmid.
  • Figure 3 (A) Southern blot analysis of the acceptor cell lines generated byJentiviral transduction of the HILO_RMCE acceptor locus (names containing a hyphen followed by "A" and a clone number) indicates that all acceptor lines contain only one lentivirus integration site.
  • the parental cell controls have no lentiviral-specifiasequences, as expected (B)
  • the acceptor cell lines were co-transfected in a 12-well (HEK293T-A2, HeLa-A12, HeLa-S3-A6, A549-A11 , HT1080-A4, U2OS-A13, L929-A6) or 6-well format (P19-A9, CAD- A13, N2a-5A) with a mixture containing 90% of pRD1 plasmid and 10% of the Cre-encoding plasmid pCAGGS-Cre. Following the puromycin selection, surviving colonies were stained with methylene blue and photographed.
  • Figure 4 Diagram of the HILO-RMCE reaction using the pRD-RIPE donor plasmid.
  • HEK293T-A2 cells containing the RMCE acceptor locus were co-transfected in a 12-well format with mixtures containing pRD-RIPE plasmid and various amounts of Cre-encoding plasmids pCAGGS-Cre or pCAGGS-nlCre. Puromycin-resistant colonies were stained with methylene blue and photographed.
  • B Same experiment as in (A) was repeated with the HeLa-A12 acceptor cells. Graph on the right shows the number of HeLa-A12 colonies per 1 //g of pRD-RIPE donor plasmid DNA as a function of Cre plasmid concentration.
  • HILO-RMCE colonies produced by co-transfecting HEK293T-A2 and HeLa-A12 cells with pCAGGS-nlCre and either pRD1 or pRD-RIPE were pooled and incubated with 2 /vg/ml Dox for 48 hours or alternatively left untreated. The EGFP expression was then studied using fluorescence-activated cell sorting (FACS).
  • FIG. 1 HEK293T-A2 cells containing 4 different RIPE-shRNAs against the PTBP1 gene were incubated with Dox for 72 hours [Dox(+)] to induce the shRNA expression or left untreated [Dox(-)].
  • Dox(+) The efficiency of PTBP1 mRNA knock down was then assayed by RT-qPCR using HPRT mRNA levels as a normalization control.
  • the graphs show residual PTBP1 expression in the Dox(+) samples as % of the corresponding Dox(-) controls. Data are averaged from 3RT- qPCR amplifications ⁇ SD.
  • FIG. 7 Using HILO-RMCE for rapid transgenesis in mammalian cell line.
  • HEK293T-A2 cells were co-transfected with pCAGGS-nlCre and different pRD vector-based donor plasmids additionally encoding: (A) CAG promoter- driven intron-containing dTomato gene (i-dTom); (B) CAG-driven EGFP gene containing a nuclear localization sequence and preceded by an internal ribosomal entry site (IRES-nlEGFP); (C) CAG-driven bicistronic cassest containing both dTomato gene (dTom) and IRES-nlEGFP; and (D) constitutively expressed reverse tetracycline transactivator (rtTA3) gene and a tetracycline-induced tRFP gene.
  • Recombinant cells were selected for 8 days with 5 ⁇ g/m ⁇ puromycin, pooled and propagated for another 4 days.
  • FIG. 1 Flowchart of a typical HILORMCE shRNA experiment.
  • B Diagram of the HILO- RMCE reaction using the pRD1 donor plasmid.
  • C The newly established 11 acceptor lines were co-transfected in a 12-well (HEK293T-A2, HeLa-A12, HeLa- S3-A6, A549-A11 , HT1080-A4, U2OS-A13, L929-A12, NIH 3T3-A7) or 6-well format (P19-A9, CAD-A13, N2a-A5) with a mixture containing 90% of pRD1 plasmid and 10% of a Cre-encoding plasmid (most cell lines, pCAGGS-Cre; NIH 3T3-A7, pCAGGS-nlCre) or the EGFP-encoding control plasmid pCIG. Following the puromycin selection, multiple colonies formed in the presence of Cre but not when Cre was substituted
  • FIG. 9 Developing the HILO-RMCE technology.
  • A Diagram of the HILO-RMCE reaction using the pRD-RIPE donor plasmid.
  • B-C Optimization of the HILO-RMCE protocol.
  • B HEK293T-A2 cells containing the RMCE acceptor locus were co-transfected in a 12-well format with the pRD-RIPE plasmid and the indicated amounts of the pCAGGS-Cre or pCAGGS-nlCre plasmids. Puromycin-resistant colonies were stained with methylene blue and photographed.
  • C The experiment described in (B) was repeated with the HeLa- A12 acceptor line.
  • Genomic DNA was isolated from 3 parental cell lines (HEK293T, HeLa and A549; lanes labeled "parent") as well as the corresponding HILO-RMCE acceptor clones ("A” followed by the clone number) and pooled clones obtained by the HILO-RMCE-mediated integration of the RIPE cassette ("A+RIPE") and analyzed by multiplex PCR using either the 5' junction primer mixture (EF, BR, and PR; Fig.
  • HILO-RMCE colonies produced by co-transfecting HEK293T-A2 and Hel_a-A12 cells with pCAGGS-nlCre and either pRD1 or pRD-RIPE were pooled and incubated with 2 yg/ml Dox for 48 hours or left untreated. The EGFP expression was then studied using FACS. Note that nearly all cells express EGFP in the Dox-treated pRD-RIPE samples.
  • F HEK293TA2 cells carrying RIPE cassettes with shRNAs against either FLuc or LacZ were induced with Dox for 36 hours or left untreated. The cells were then transfected with a mixture of plasmids encoding the FLuc and RLuc luciferases and the FLuc activities normalized to the corresponding RLuc signals. Data are averaged from 6 transfection experiments ⁇ SD.
  • HEK293T-A2 cells containing four different RIPE-encoded shRNAs against human PTBP1 mRNA or the shFLuc shRNA were induced with Dox for 72 hours and the efficiency of the PTBP1 knockdown analyzed by reverse transcription- quantitative PCR (RT-qPCR) (top) and immunoblotting with PTBP1 -specific antibodies (bottom).
  • the RT-qPCR graph shows relative PTBP1 expression levels normalized to the shFLuc control.
  • anti-GAPDH antibody was used to control lane loading.
  • B-C The experiment in (A) was repeated in N2a-A5 cells using shRNAs against mouse PTBP1 (B) or Ago2 (C) mRNAs. Note that co-expressing the two most potent Ago2-specific shRNAs from a single RIPE cassette further improves the protein knockdown (lane "sh1 +sh4" in C).
  • FIG. 11 Using HILO-RMCE to knock down the human TUT family members.
  • A An shRNA library against the human TUT family was integrated into HEK293T-A2 cells and the knockdown efficiencies determined by RT-qPCR. The TUT expression levels in the Dox-treated samples are normalized to the corresponding Dox-negative controls.
  • B The efficiencies of the TUT4- specific shRNAs were also studied by immunoblotting using anti-TUT4 antibody.
  • C RT-qPCR analysis showing down-regulation of TUT2/GLD2 and
  • TUT4/ZCCHC11 by corresponding shRNAs in three additional human acceptor lines Hel_a-A12, A549-1 1 , U20S- 13. Expression levels are normalized to the Dox-treated shFLuc controls as in (Fig. 3). In all RT-qPCR graphs, data are averaged from three amplifications experiments ⁇ SD.
  • HEK293T-A2 cells were co-transfected with pCAGGS-nlCre and pRD1 -based donor plasmid (pEM705) containing a CAG promoter-driven bicistronic cassette encoding dTomato (dTom) (34) and a nuclear localized EGFP proteins. Recombinant cells were selected with
  • FIG. 13 Southern blot analysis of the human and mouse acceptor cell lines generated by lentiviral transduction of the HILO-RMCE acceptor locus (lanes marked with "A” and the clone number) were carried out using /Vcol-cut genomic DNAs and a lentivirus vector-specific 32Plabeled probe. The presence of a single band in the acceptor line samples corresponds to a single lentiviral integration site within the cellular genome. The parental cells contain no vectorspecific sequences, as expected.
  • NLS-containing Cre is a more efficient HILO-RMCE recombinase than the wildtype Cre.
  • L929-A12 cells containing the RMCE acceptor locus were co-transfected in a 12-well format with the pRD-RIPE plasmid and the indicated amounts of the pCAGGS-Cre or pCAGGSnlCre plasmids. Puromycin-resistant colonies were stained with methylene blue and photographed. Note that the well corresponding to the optimal pCAGGS-nlCre concentration (2.5%) contains noticeably larger number of colonies than the well with the optimal pCAGGSCre concentration (1 %). [0049].
  • the acceptor locus is uniformly rearranged in HILO- RMCE-generated cell pools.
  • Genomic DNA was isolated from 3 parental cell lines (HEK293T, HeLa and A549; lanes labeled "parent"), the corresponding HILO-RMCE acceptor clones ("A” followed by the clone number) and pooled clones obtained by the HILO-RMCE-mediated integration of the RIPE cassette (the "A+RIPE" lanes).
  • the DNA samples were analyzed by Southern blotting as in the Fig. 3A to confirm the uniform rearrangement of the acceptor locus as a result of the RMCE reaction.
  • the data are consistent with the expected increase in the length of the acceptor locus-specific Nco ⁇ fragment by 856 bp following the RIPE integration.
  • Figure 16 Uniform induction of EGFP expression in HILO-RMCE cell pools.
  • HEK293T-A2 and Hel_a-A12 cell pools containing the HILO-RMCE- integrated RIPE cassette were incubated with 2 /vg/ml doxycycline for 48 hours or alternatively left untreated.
  • the EGFP expression was then studied using epifluorescence and phase contrast (PhC) microscopy. Note that all viable cells express EGFP in doxycycline-treated samples, whereas EGFP is not detected in the corresponding doxycycline-negative controls.
  • HILO-RMCE generates virtually homogeneous cell populations expressing doxyciclin-inducible shRNAs.
  • A Genomic DNAs from the parental HEK293T line, the acceptor HEK293T-A2 line, and pooled HILO-RMCE clones containing RIPE-encoded shFLuc or shLacZ shRNAs were analyzed by multiplex PCR to detect the changes at the 5' (primers EF, BR, and PR; see Fig. 2A and Table 1 ) and the 3' boundaries (GF, BF, and WR; Fig. 2A and Table 1 ) of the acceptor locus following the integration of the RIPE cassettes.
  • FIG. 1 Knocking down Ptbpl by shRNAs changes alternative splicing patterns of the Ptbpl target genes.
  • CAD-A13 cells containing RIPE- encoded shRNAs against mouse Ptbpl (sh2 or sh4) or the shFLuc shRNA were induced with 2 jc/g/ml doxycycline for 72 hours.
  • RT-qPCR we used the previously described RT-PCR procedure (36) to analyze the splicing patterns of three alternative cassette exons known to be repressed by the Ptbpl protein: exon 10 of the Ptbpl gene, exon N1 of the Src gene and exon 5 of the Cltb gene.
  • HEK293T-A2 cell pools containing four different RIPE-encoded shRNAs against human TUT6 mRNA or the shFLuc shRNA were induced with Dox for 72 hours or left untreated and the TUT6 knockdown efficiency was analyzed by RT-qPCR.
  • the expression levels in the Dox-treated samples are normalized to the corresponding Dox-negative controls. Data are averaged from three
  • FIG. 20 Using HILO-RMCE for engineering of transgenic cell populations.
  • HEK293T-A2 cells were co-transfected with pCAGGS-nlCre and either of the two pRD vector-based donor plasmids encoding (A) CAG-driven EGFP gene containing a nuclear localization sequence and preceded by an internal ribosomal entry site (IRES-nlEGFP) or (B) CAG promoter-driven introncontaining dTomato gene (i-dTom).
  • Recombinant cells were selected with puromyein for 7 days, pooled and propagated for another 4 days. Images were taken using phase contrast and appropriate epifluorescence filters. Maps of the corresponding donor constructs (pEM652 and pEM689) are shown on the top of the panels
  • HILO high-efficiency and low-background recombination-mediated cassette exchange
  • a cell includes one or more cells and equivalents thereof known to those skilled in the art.
  • a " targeting nucleic acid site” comprises a nucleotide sequence flanked by two non-identical recombination sites.
  • a targeting nucleic acid site targets or provides a "specific chromosomal site”.
  • a "transfer cassette” for use with a given target site comprises a nucleotide sequence flanked by the same two non-identical recombination sites present in the corresponding target site.
  • the terms “transfer cassette”, “donor cassette” and “targeting cassette” are used interchangeably herein.
  • a target site and a transfer cassette may each comprise more than two non-identical recombination sites.
  • a "donor construct” is a recombinant construct that contains a transfer cassette.
  • the terms “donor construct” and “donor vector” are used
  • Transgenic refers to any cell, cell line, the genome of which has been altered by the presence of a heterologous nucleic acid, such as a recombinant DNA construct, including those initial transgenic events.
  • the term "transgenic” as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non- recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
  • Gene as it applies to cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components (e.g., mitochondrial) of the cell.
  • Heterologous with respect to sequence means a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • nucleic acid sequence is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non- natural or altered nucleotide bases.
  • Nucleotides are referred to by their single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uhdylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), "K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.
  • Polypeptide “peptide”, “amino acid sequence” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • polypeptide “peptide”, “amino acid sequence”, and “protein” are also inclusive of
  • glycosylation lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.
  • mRNA essential RNA
  • cDNA refers to a DNA that is complementary to and synthesized from a mRNA template using the enzyme reverse transcriptase.
  • the cDNA can be single-stranded or converted into the double-stranded form using the Klenow fragment of DNA polymerase I.
  • “Mature” protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or pro-peptides present in the primary translation product have been removed.
  • Precursor protein refers to the primary product of translation of mRNA; i.e., with pre- and pro-peptides still present. Pre- and propeptides may be and are not limited to intracellular localization signals.
  • isolated refers to materials, such as nucleic acid molecules and/or proteins, which are substantially free or otherwise removed from components that normally accompany or interact with the materials in a naturally occurring environment. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized
  • Recombinant refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the
  • Recombinant also includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or a cell derived from a cell so modified, but does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.
  • naturally occurring events e.g., spontaneous mutation, natural transformation/transduction/transposition
  • Recombinant DNA construct refers to a combination of nucleic acid fragments that are not normally found together in nature. Accordingly, a
  • recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that normally found in nature.
  • regulatory sequences refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence.
  • Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
  • promoters refers to a nucleic acid fragment capable of controlling transcription of another nucleic acid fragment.
  • Promoter functional in a cell is a promoter capable of controlling transcription in cells whether or not its origin is from that cell.
  • operably linked refers to the association of nucleic acid fragments in a single fragment so that the function of one is regulated by the other.
  • a promoter is operably linked with a nucleic acid fragment when it is capable of regulating the transcription of that nucleic acid fragment.
  • expression refers to the production of a functional product.
  • expression of a nucleic acid fragment may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or functional RNA) and/or translation of mRNA into a precursor or mature protein.
  • “Introduced” in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct) into a cell means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic cell where the nucleic acid fragment may be
  • transfected mRNA incorporated into the genome of the cell (e.g., chromosome, plasmid), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
  • a "transformed cell” is any cell into which a nucleic acid fragment (e.g., a recombinant DNA construct) has been introduced.
  • Transformation refers to both stable transformation and transient transformation.
  • “Stable transformation” refers to the introduction of a nucleic acid fragment into a genome of a host organism resulting in genetically stable inheritance. Once stably transformed, the nucleic acid fragment is stably integrated in the genome of the host organism and any subsequent generation.
  • Transient transformation refers to the introduction of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without genetically stable inheritance.
  • Selection agent refers to a compound which is toxic to non- transformed cells and which kills non-transformed cells when it is incorporated in the culture medium in an "effective amount", i.e., an amount equal to or greater than the minimal amount necessary to kill non-transformed cells.
  • Cells can be transformed with an appropriate gene, such that expression of that transgene confers resistance to the corresponding selection agent, via de-toxification or another mechanism, so that these cells continue to grow and are subsequently able to regenerate cells.
  • the gene conferring resistance to the selection agent is termed the "selectable marker gene", "selectable marker” or “resistance gene”.
  • Transgenic cells that lack a functional selectable marker gene will be killed by the selection agent.
  • Selectable marker genes include genes conferring resistance to antibiotics, herbicidal compounds.
  • a specific selection agent may have one or more corresponding selectable marker genes.
  • a specific selectable marker gene may have one or more corresponding selection agents.
  • Suitable selection agents include but are not limited to, cytotoxic agents such as blasticidin, puromycin, hygromycin, sulfonylurea herbicides such as chlorsulfuron, nicosulfuron and hmsulfuron, and other herbicides which act by inhibition of the enzyme acetolactate synthase (ALS), glyphosate, bialaphos and phosphinothricin (PPT). It is also possible to use positive selection marker systems such as phospho-mannose isomerase and similar systems which confer positive growth advantage to transgenic cells.
  • FIG 1 depicts a flowchart of the HILO procedure.
  • a lentiviral vector-encoded cassette consisting of a strong constitutive promoter from the EFIagene and a downstream blasticidin resistance gene (Bsd) flanked ("floxed") by mutually incompatible Cre recombinase-specific sites Lox2272 and LoxP (see Figure 2).
  • the blasticidin-resistant acceptor cell lines were co-transfected in a 12- or 6- well format with a mixture of pRD1 and either a plasmid encoding wild type Cre recombinase under the strong constitutive promoter CAG (pCAGGS-Cre) or a control plasmid encoding a CAG-driven EGFP gene (pCIG).
  • pCAGGS-Cre strong constitutive promoter CAG
  • pCIG control plasmid encoding a CAG-driven EGFP gene
  • RNAi performance of the RIPE cassette we inserted a firefly luciferase (FLuc) specific shRNA (shFLuc) at the pre-miR-155 cloning site and generated a pool of HEK293T-A2 cells carrying the RIPE-shFLuc cassette using HILO-RMCE.
  • the cells were either pre-incubated with Dox [Dox(+)] or left untreated [Dox(-)].
  • the two populations were then transfected with a mixture of plasmids encoding FLuc and Renilla luciferase (RLuc) and the relative FLuc expression measured as a ratio between the FLuc and the RLuc activities.
  • TUTases human terminal uridyl transferases
  • HILO-RMCE allows one to induce stable shRNA-medicated RNAi in mammalian cells with ease and penetrance previously only possible using siRNA. Since different shRNA cassettes inserted at the same acceptor locus have comparable expression levels, this approach will substantially accelerate experimental validation and scoring of computationally designed sh RNAs. The cost efficiency and the technical simplicity of HILO-RMCE should make it a popular alternative to the retro and lentivirus vector-based high-throughput shRNA screens. Our data on the other types of transgenic cassettes (see figure 7) suggests that the method will facilitate a wide range of molecular and cell biology experiments by providing a possibility of rapid integration of transgenic sequence into a cell line of interest.
  • the process allows yields of virtually homogeneous cell populations of genetically altered eukaryotic cells in a rapid and cost-effective manner.
  • the new process comprised the steps of (a) delivering a nucleic acid sequence of interest operably linked with site-specific recombination sites into a eukaryotic cell; (b) integrating the sequence of interest at a predefined genetic locus through the activity of a site-specific recombinase and (c) obtaining nearly homogenous populations of cells with a correctly integrated nucleic acid sequence of interest using s selection procedure that does not require substantioal human
  • a eukaryotic cell is a mammalian cell.
  • the term operably linked refers to a juxtaposition of components permitting them to function in an intended manner.
  • the term nearly homogenous means preferably 90% to 100% homogenous, this may be 90% homogenous, 95% homogenous, 97% homogenous, 99% homogenous, or 100% homogenous.
  • reagents that comprise nucleic acid constructs with a site-specific recombination site and a site specific recombinase.
  • the reagent may further comprise a nucleic acid sequence of interest.
  • homogenous populations of genetically identical clones that include a nucleic acid sequence of interest may also fall within the scope of the invention when they are obtained by the process disclosed.
  • the process described is advantageous to transgenic technologies as it immediately yields nearly homogenous populations of genetically identical clones thus bypassing laborious cell cloning and enrichment steps commonly known in the field.
  • the improvement is made possible through the combination of rationally designed genetic constructs and proprietary cell lines derived in our laboratory from parental cell lines by integrating a single copy of an acceptor cassette containing a eukaryotic promoter and a selection marker flanked by two site-specific recombination sites, which was followed by selecting cell clones with properties advantageous for the described art.
  • the process offers considerable savings in terms of time, resources and human effort.
  • the reagents can be used in the process for academic and commercial institutions performing molecular and cell biology work in eukaryotic cells. This includes but is not limited to RNAi experiments, gene overexpression studies, cell engineering and synthetic biology project. While it is primarily designed as a research and development tool it may also be used to create therapeutic constructs in vitro for use in gene therapy.
  • HILO high-efficiency and low-background
  • Fig. 8A To establish acceptor lines compatible with the HILO-RMCE procedure, we assembled a lentiviral vector-encoded cassette containing the human EF-1a promoter and a blasticidin resistance gene (Bsd) "floxed" by the mutually incompatible Cre recombinase specific sites Lox2272 and LoxP (Fig. 8B). A single copy of this cassette was transduced into 6 human (HEK293T, HeLa, HeLa-S3, A549, HT10 .
  • Bsd blasticidin resistance gene
  • TK pA polyadenylation signal
  • the acceptor cell lines were co-transfected with a mixture of pRD1 and either the pCAGGS-Cre plasmid encoding a wild-type Cre recombinase or a control plasmid encoding EGFP (pCIG). In all cases, multiple colonies appeared 5-10 days following puromycin selection in the presence of Cre but not in the control wells expressing EGFP (Fig. 8C). Thus, the HILO-RMCE reaction proceeded efficiently and with a negligibly low background of Cre-independent integration, as intended.
  • RNAi firefly luciferase (FLuc) specific shRNA (shFLuc) at the pre-miR-155 cloning site and integrated the RIPEshFLuc cassette into the HEK293T-A2 cells using HILO- RMCE (Fig. 17).
  • the cells were either pre-incubated with Dox or left untreated.
  • the two populations were then transfected with a mixture of plasmids encoding FLuc and Renilla luciferase (RLuc) and the relative FLuc expression measured as a ratio between the Flue and the Rluc activities (Fig. 9F).
  • LacZ-specific shRNA control shLacZ
  • Fig. 9F LacZ-specific shRNA control
  • TUTs human terminal uridyl transferases
  • the screen identified at least one potent shRNA for each TUT gene that substantially reduced the target expression in a Dox-dependent manner (Fig. 4A and Fig. 19).
  • the efficiency of the TUT4-specific shRNAs was further confirmed using immunoblotting (Fig. 1 1 B).
  • the optimized shRNAs against TUT2/GLD2 and TUT4/ZCCHC1 1 the two TUTs involved in the microRNA pathway (25, 26), fared well in other human acceptor lines (Fig. 1 1 C).
  • the sh3 TUT4/ZCCHC1 shRNA outperformed a previously published siRNA (25) and a commercially available siRNA mixture (ONTARGETplus, Dharmacon; not shown).
  • HILO-RMCE transforms the shRNA experiment into a user-friendly procedure that combines the possibility of long-term RNAi with the speed and convenience of siRNA-based approaches.
  • the cost efficiency and the technical simplicity make HILO-RMCE an ideal platform for optimizing shRNA efficiency and other types of low-throughput projects in diverse laboratory settings, as well as a useful addition to the high-throughput RNAi screening toolbox.
  • our data on other types of transgenic cassettes suggest that the described platform should facilitate a wider range of molecular and cell biology experiments.
  • RMCE acceptor lentiviral vector pEM584.
  • pHAGE backbone 2-7 by inserting a human EF-1a promoter from a derivative of the pEF-BOS vector (28) followed by a Lox2272- and LoxP-floxed blasticidin resistance gene (Bsd) from the pLenti6A/5-DEST plasmid (Invitrogen) between the cpPu ⁇ Trip) and WPRE elements.
  • the RMCE donor vector (pRD1 ; alternative name ⁇ ) was generated by subcloning the polyadenylation signal-containing HSV thymidine kinase (TK) gene from a pEasyFlox derivative (29) into the pBluescript II KS(+) backbone. Immediately downstream of the TK polyadenylation signal, we introduced a Lox2272- and LoxP-floxed promoter-less puromycin resistance ⁇ Pur) gene from the pPUR plasmid (Clontech).
  • TK polyadenylation signal-containing HSV thymidine kinase
  • pRD-RIPE plasmid (alternative name pEM791 ) was derived from pRD1 by removing the TK promoter and coding sequences and inserting a UBC promoter-driven reverse tetracycline transactivator gene ⁇ rtTA3) and the tet-inducible promoter TRE (both elements adapted from the pTRIPZ plasmid, Open Biosystems) between the Pur gene and the LoxP site.
  • the TRE promoter was followed by an intron-EGFP module containing an intronic pre-miR-155-based shRNA cloning site with two BsmBl sequences (30).
  • pCAGGS-nlCre (alternative name pEM784) was modified from pCAGGS-Cre (31 ) by substituting the original sequence 5'- ACTTTACTTAAAACCATTATCTGAGTGTGAAATG-3' in front of the Cre gene with the sequence 5'-CTAGACTCGACCATGCCCAAGAAGAAGAGGAAGGTG-3' encoding the N-terminal nuclear localization sequence (NLS; underlined) from the Large T antigen of the SV40 virus. Complete sequences and maps of all pEM plasmids are available upon request.
  • shRNAs were designed using the miR RNAi design option of the Block- iT RNAi Designer program (https://rnaidesigner.invitrogen.com/rnaiexpress/). Wherever possible, multiple shRNAs targeting the open reading frame (ORF) and 3' untranslated region (3'UTR) were chosen for a given target gene.
  • ORF open reading frame
  • 3'UTR 3' untranslated region
  • shRNA For each shRNA, two 64 nt long complementary DNA oligonucleotides were ordered from Sigma Life Science, annealed and inserted into pRD-RIPE plasmid at the BsmBI sites (for detailed description of the pre-miR-155 based shRNA cloning strategy, see (30) and https://rnaidesigner.invitrogen.com/rnaiexpress/).
  • shFLuc the firefly liciferase-specific shRNA (shFLuc) was engineered by annealing the following oligonucleotides: SEQ ID NO 1 : 5'-
  • the mAgo2 sh4 shRNA element was amplified using the RIPE-specific primers shRNAdimer_F1 and shRNAdimer_R1 (Table 1 ), the PCR fragment was treated with Mfe ⁇ (NEB; underlined sequence) and inserted into the mAgo2 sh1 -containing RIPE cassette at the EcoRI-EcoRV sites located downstream of the miR 0 155 element to create an intron-encoded sh1- sh4 tandem. Since the shRNAdimer_R1 primer restores both the EcoRI and the EcoRV sites (italicized sequences), this procedure can theoretically be repeated to generate tandem shRNA arrays of any desired length and complexity.
  • hTUTasel _shRNA1_ CCTGTAAAGTGCCACAGTATCAGGTCAGTCAGTGGCCAAAACCTGATACTCAGTGGCA
  • hTUTasel _shRNA2_ CCTGTTTGATATCAGCCAACCCAGTCAGTCAGTGGCCAAAACTGGGTTGGTACTGATAT
  • hTUTasel _shRNA3_ CCTGCAAGGTGGAATTCCCATGCGTCAGTCAGTGGCCAAAACGCATGGGAGAATTCCA
  • hTUTasel _shRNA4_ TGCTGAAGAAACGTGATCTGAATGGAGTTTTGGCCACTGACTGACTCCATTCATCACGT
  • hTUTasel _shRNA4_ CCTGAAGAAACGTGATGAATGGAGTCAGTCAGTGGCCAAAACTCCATTCAGATCACGT
  • hTUTasel _shRNA5_ CCTGCAAAGGGTCTGTATGCAGTGTCAGTCAGTGGCCAAAACACTGCATAGTCAGACC
  • hTUTasel _shRNA6_ CCTGAACACAGACAGTCTTCTGAGTCAGTCAGTGGCCAAAACTCAGAAGATCCTGTCT
  • hTUTase2 _shRNA1_ CCTGTTGTTGATGTTGGAGTGGTCAGTCAGTGGCCAAAACCACTCCAAATCATCAAC
  • hTUTase2 _shRNA3_ CCTGAAATAACTCCAATCTGCTGGTCAGTCAGTGGCCAAAACCAGCAGATACTGGAGT
  • hTUTase2 _shRNA4_ CCTGATCACCAGCACACGGACGAGTCAGTCAGTGGCCAAAACTCGTCCGTTAGTGCTG
  • hTUTase2 _shRNA5_ TGCTGATTACATGGAGCTTGATGTACGTTTTGGCCACTGACTGACGTACATCACTCCAT
  • hTUTase2 _shRNA5_ CCTGATTACATGGAGTGATGTACGTCAGTCAGTGGCCAAAACGTACATCAAGCTCCAT
  • hTUTase2 shRNA6 CCTGTACACATCTCTAGGATTAGGTCAGTCAGTGGCCAAAACCTAATCCTGAAGAGATG
  • hTUTase2 shRNA7 TGCTGAGTATTTCATGACTGCAGTGAGTTTTGGCCACTGACTGACTCACTGCACATGAA
  • hTUTase2 shRNA7 CCTGAGTATTTCATGTGCAGTGAGTCAGTCAGTGGCCAAAACTCACTGCAGTCATGAAA
  • hTUTase3 shRNAI TGCTGATCAAAGGCCTGCTTCACTTGGTTTTGGCCACTGACTGACCAAGTGAAAGGCC
  • hTUTase3 shRNAI CCTGATCAAAGGCCTTTCACTTGGTCAGTCAGTGGCCAAAACCAAGTGAAGCAGGCCT
  • hTUTase3 shRNA2 TGCTGATATGTGGCAACTTCATCTGTGTTTTGGCCACTGACTGACACAGATGATTGCCA
  • hTUTase3 shRNA2 CCTGATATGTGGCAATCATCTGTGTCAGTCAGTGGCCAAAACACAGATGAAGTTGCCA
  • hTUTase3 shRNA3 TGCTGATTGCATGAAGGCTCAGGTCTGTTTTGGCCACTGACTGACAGACCTGACTTCAT
  • hTUTase3 shRNA3 CCTGATTGCATGAAGTCAGGTCTGTCAGTCAGTGGCCAAAACAGACCTGAGCCTTCAT
  • hTUTase3 shRNA4 TGCTGTTGAGTTGTACCTTGGAAGCCGTTTTGGCCACTGACTGACGGCTTCCAGTACA
  • hTUTase3 shRNA4 CCTGTTGAGTTGTACTGGAAGCCGTCAGTCAGTGGCCAAAACGGCTTCCAAGGTACAA
  • hTUTase3 shRNA5 TGCTGAACAGAGCCATACACATTGATGTTTTGGCCACTGACTGACATCAATGTATGGCT
  • hTUTase3 shRNA5 CCTGAACAGAGCCATACATTGATGTCAGTCAGTGGCCAAAACATCAATGTGTATGGCTC
  • hTUTase3 shRNA6 TGCTGTTCACAGTCACCTTAAAGCTGGTTTTGGCCACTGACTGACCAGCTTTAGTGACT
  • hTUTase3 shRNA6 CCTGTTCACAGTCACTAAAGCTGGTCAGTCAGTGGCCAAAACCAGCTTTAAGGTGACT
  • hTUTase3 shRNA7 TGCTGTAATAACTACACCTCCTAGTCGTTTTGGCCACTGACTGACGACTAGGATGTAGT
  • hTUTase3 shRNA7 CCTGTAATAACTACATCCTAGTCGTCAGTCAGTGGCCAAAACGACTAGGAGGTGTAGTT
  • hTUTase4 shRNAI CCTGATACCCTTGAGAGCAGGATGTCAGTCAGTGGCCAAAACATCCTGCTGTCTCAAG
  • hTUTase4 shRNA2 TGCTGTTCAGAGCAAATCTAGTCAGAGTTTTGGCCACTGACTGACTCTGACTATTTGCT
  • hTUTase4 shRNA2 CCTGTTCAGAGCAAATAGTCAGAGTCAGTCAGTGGCCAAAACTCTGACTAGATTTGCTC
  • hTUTase4 shRNA3 CCTGTAAACCAGCTGTGTTTAAGGTCAGTCAGTGGCCAAAACCTTAAACAGCCAGCTG
  • hTUTase4 shRNA4 TGCTGAAGAGATGAAGAGTCCTGTCCGTTTTGGCCACTGACTGACGGACAGGACTTCA
  • hTUTase4 shRNA4 CCTGAAGAGATGAAGTCCTGTCCGTCAGTCAGTGGCCAAAACGGACAGGACTCTTCAT
  • hTUTase4 shRNA5 TGCTGTGCAGATGCTGCATACTATCGGTTTTGGCCACTGACTGACCGATAGTACAGCA
  • hTUTase4 shRNA5 CCTGTGCAGATGCTGTACTATCGGTCAGTCAGTGGCCAAAACCGATAGTATGCAGCAT
  • hTUTase4 shRNA6 TGCTGATCAATAGCTGCATAAGTAGCGTTTTGGCCACTGACTGACGCTACTTACAGCTA
  • hTUTase4 shRNA6 CCTGATCAATAGCTGTAAGTAGCGTCAGTCAGTGGCCAAAACGCTACTTATGCAGCTAT
  • hTUTase4 shRNA7 TGCTGAATAGCAGCTGACTGGGAAGAGTTTTGGCCACTGACTGACTCTTCCCACAGCT
  • hTUTase4 shRNA7 CCTGAATAGCAGCTGTGGGAAGAGTCAGTCAGTGGCCAAAACTCTTCCCAGTCAGCTG
  • hTUTase5 shRNAI CCTGTAAAGGAGGACTCCCATTTGTCAGTCAGTGGCCAAAACAAATGGGAGCGTCCTC
  • hTUTase5 shRNA2 TGCTGTTGTCAAGGACTTTGATGGAAGTTTTGGCCACTGACTGACTTCCATCAGTCCTT
  • hTUTase5 shRNA3 TGCTGAATTCTTGATGAACTCCGCTGGTTTTGGCCACTGACTGACCAGCGGAGCATCA
  • hTUTase5 shRNA3 CCTGAATTCTTGATGCTCCGCTGGTCAGTCAGTGGCCAAAACCAGCGGAGTTCATCAA
  • hTUTase5 shRNA4 TGCTGAACTGTAGAAAGCTAATGGCCGTTTTGGCCACTGACTGACGGCCATTATTTCTA
  • hTUTase5 shRNA4 CCTGAACTGTAGAAATAATGGCCGTCAGTCAGTGGCCAAAACGGCCATTAGCTTTCTA
  • hTUTase5 shRNA5 TGCTGAACAATGACCTCCCAAGCTTAGTTTTGGCCACTGACTGACTAAGCTTGAGGTCA
  • hTUTase5 shRNA5 CCTGAACAATGACCTCAAGCTTAGTCAGTCAGTGGCCAAAACTAAGCTTGGGAGGTCA
  • hTUTase5 shRNA6 TGCTGTATACCTGTATGCTTCCGACGGTTTTGGCCACTGACTGACCGTCGGAAATACA
  • hTUTase5 shRNA6 CCTGTATACCTGTATTTCCGACGGTCAGTCAGTGGCCAAAACCGTCGGAAGCATACAG
  • hTUTase5 shRNA7 TGCTGATAACTTTCGGTCATCGAGAAGTTTTGGCCACTGACTGACTTCTCGATCCGAAA
  • hTUTase5 shRNA7 CCTGATAACTTTCGGATCGAGAAGTCAGTCAGTGGCCAAAACTTCTCGATGACCGAAA
  • hTUTase6 shRNA TGCTGCTCGGAAGCAACTTCCGCCGAGTTTTGGCCACTGACTGACTCGGCGGATTGCT
  • hTUTase6 shRNAI CCTGCTCGGAAGCAATCCGCCGAGTCAGTCAGTGGCCAAAACTCGGCGGAAGTTGCT
  • hTUTase6 shRNA2 TGCTGACACAAGCACCTCTGCCACCAGTTTTGGCCACTGACTGACTGGTGGCAGGTGC
  • hTUTase6 shRNA2 CCTGACACAAGCACCTGCCACCAGTCAGTCAGTGGCCAAAACTGGTGGCAGAGGTGC
  • hTUTase6 shRNA3 TGCTGATGCTAGGAAGTACTCAGAGAGTTTTGGCCACTGACTGACTCTCTGAGCTTCCT
  • hTUTase6 shRNA3 CCTGATGCTAGGAAGCTCAGAGAGTCAGTCAGTGGCCAAAACTCTCTGAGTACTTCCT
  • hTUTase6 shRNA4 TGCTGAGAAGAGGTCAAGATCACAGCGTTTTGGCCACTGACTGACGCTGTGATTGACC
  • hTUTase6 shRNA4 CCTGAGAAGAGGTCAATCACAGCGTCAGTCAGTGGCCAAAACGCTGTGATCTTGACCT
  • hTUTase6 shRNA5 CCTGCCCACAAGCTTTCATTTGTGTCAGTCAGTGGCCAAAACACAAATGATAAAGCTTG
  • hTUTase6 shRNA6 TGCTGAGAACTTGACCACAGGGCGCCGTTTTGGCCACTGACTGACGGCGCCCTGGTC
  • hTUTase6 shRNA6 CCTGAGAACTTGACCAGGGCGCCGTCAGTCAGTGGCCAAAACGGCGCCCTGTGGTCA
  • hTUTase7 shRNAI CCTGAGTTGAAGCCTTCTACACTGTCAGTCAGTGGCCAAAACAGTGTAGATCAGGCTT
  • hTUTase7 shRNA2 TGCTGATAACAGGAATTCTAGGTCCAGTTTTGGCCACTGACTGACTGGACCTAATTCCT
  • hTUTase7 shRNA2 CCTGATAACAGGAATTAGGTCCAGTCAGTCAGTGGCCAAAACTGGACCTAGAATTCCT
  • hTUTase7 shRNA3 TGCTGTTGAAACCCAATCTGCTACAGGTTTTGGCCACTGACTGACCTGTAGCATTGGGT
  • hTUTase7 shRNA3 CCTGTTGAAACCCAATGCTACAGGTCAGTCAGTGGCCAAAACCTGTAGCAGATTGGGT
  • hTUTase7 shRNA4 TGCTGAACACAGGTTGACTATTTAGGGTTTTGGCCACTGACTGACCCTAAATACAACCT
  • hTUTase7 shRNA4 CCTGAACACAGGTTGTATTTAGGGTCAGTCAGTGGCCAAAACCCTAAATAGTCAACCTG
  • hTUTase7 shRNA5 TGCTGTAAAGTGGCAAGTCCCTCACAGTTTTGGCCACTGACTGACTGTGAGGGTTGCC
  • hTUTase7 shRNA5 CCTGTAAAGTGGCAACCCTCACAGTCAGTCAGTGGCCAAAACTGTGAGGGACTTGCCA
  • hTUTase7 shRNA6 TGCTGTACCGTAGGAGACTTGCCTTTGTTTTGGCCACTGACTGACAAAGGCAACTCCTA
  • hTUTase7 shRNA6 CCTGTACCGTAGGAGTTGCCTTTGTCAGTCAGTGGCCAAAACAAAGGCAAGTCTCCTA
  • hTUTase7 shRNA7 TGCTGTTTAGTTCCTGGAAAGTCCTGGTTTTGGCCACTGACTGACCAGGACTTCAGGA
  • hPTBPI shRNAI F TGCTGTTCTCTGGAATGATGGAAGTTGTTTTGGCCACTGACTGACAACTTCCAATTCCA
  • hPTBPI shRNAI R CCTGTTCTCTGGAATTGGAAGTTGTCAGTCAGTGGCCAAAACAACTTCCATCATTCCAG
  • hPTBPI shRNA2 F TGC ' I G GTCATTTCCGTTTGCTGCAGGTT I TGGCCAC rGACTGACCTGCAGCACGGAA
  • hPTBPI shRNA2 R CCTGTGTCATTTCCGTGCTGCAGGTCAGTCAGTGGCCAAAACCTGCAGCAAACGGAAA
  • hPTBPI shRNA3 F TGCTGTATTGAACAGGATCTTCACGCGTTTTGGCCACTGACTGACGCGTGAAGCCTGT
  • hPTBPI shRNA3 R CCTGTATTGAACAGGCTTCACGCGTCAGTCAGTGGCCAAAACGCGTGAAGATCCTGTT
  • hPTBPI shRNA4 F TGCTGAATATTGCTAGGCACAGACGTGTTTTGGCCACTGACTGACACGTCTGTCTAGC
  • hPTBPI shRNA4 R CCTGAATATTGCTAGACAGACGTGTCAGTCAGTGGCCAAAACACGTCTGTGCCTAGCA
  • mPTBPI shRNAI F TGCTGAGCATGAGAAGGTTGGTAACCGTTTTGGCCACTGACTGACGGTTACCACTTCT
  • mPTBPI shRNAI R CCTGAGCATGAGAAGTGGTAACCGTCAGTCAGTGGCCAAAACGGTTACCAACCTTCTC
  • mPTBPI shRNA2 F TGCTGTCCACAATGATCCTGAGCACTGTTTTGGCCACTGACTGACAGTGCTCAATCATT
  • mPTBPI shRNA2 R CCTGTCCACAATGATTGAGCACTGTCAGTCAGTGGCCAAAACAGTGCTCAGGATCATT
  • mPTBPI shRNA3 F TGCTGATGTATAGGCCACCTGGCTCAGTTTTGGCCACTGACTGACTGAGCCAGGGCCT
  • mPTBPI shRNA3 R CCTGATGTATAGGCCCTGGCTCAGTCAGTCAGTGGCCAAAACTGAGCCAGGTGGCCTA
  • mPTBPI shRNA4 F TGCTGATACAAAGGTCACAATGAGGCGTTTTGGCCACTGACTGACGCCTCATTGACCT
  • mPTBPI shRNA4 R CCTGATACAAAGGTCAATGAGGCGTCAGTCAGTGGCCAAAACGCCTCATTGTGACCTT
  • RMCE acceptor cell lines -40% confluent cell cultures were incubated with serially dilutions of the pEM584 lentiviral stock (1 to 200 cfu per 10 cm plate) for 18 hours without polybrene. The medium was then changed and the cells were incubated for another 18 hours prior to the addition of blasticidin S to 5-10 /g/ml. The incubation was continued in the presence of blasticidin until non-infected cells died and visible blasticidin-resistant colonies formed. For each cell line, 2- 8 individual colonies were picked using 200 ⁇ pipette tips and clonally expanded. Dishes containing >50 colonies were discarded to avoid multiple integration events and colony cross-contamination. Clones with optimal RMCE performance were maintained in the presence of 2.5- 5 g/ml blasticidin S. For long-term storage, cells were cryo-preserved in a mixture containing 90% of the appropriate culture medium and 10% DMSO.
  • RMCE acceptor cell lines were plated in 12-well plates at 1.0-1.5x105 cells per well in an antibiotic-free medium 12-18 hours prior to transfection except L929-A12 and P 9-A9 cells that were plated 3 hours before transfection. Cells were then co-transfected with an RMCE donor plasmid (pRD, pRD-RIPE or a derivative of pRD-RIPE containing a gene-specific shRNA sequence) blended with 0.5-10% (w/w) of a Cre-encoding plasmid (pCAGGS-Cre or pCAGGSnlCre).
  • pRD RMCE donor plasmid
  • pRD-RIPE or a derivative of pRD-RIPE containing a gene-specific shRNA sequence
  • the maximal concentration Hel_a-A12, 2 /yg/ml; A549-A1 1 , CAD-A13, HEK293T-A2, Hel_a-S3- A6, HT1080-A4, N2a-A5, NIH 3T3-A7 and U2OS-A13, 5 yg/ml
  • L929-A12 and P19-A9 cells were immediately exposed to the maximal puromycin concentration (L929-A12, 16 vg/ml; P19-A9, 5 /yg/ml) followed by the 1 ⁇ 2 concentration step after the death of the puromycin-sensitive cells.
  • the cultures were incubated until the appearance of visible puromycin-resistant colonies, which were either pooled and expanded in a medium containing 1/2 maximal puromycin concentration or alternatively stained with 0.1 % methylene blue in 50% methanol and photographed.
  • HEK293T-A2 cells encoding RIPE-shRNAs against firefly luciferase or LacZ were pre-treated with 2 /vg/ml doxycycline for 36 hours and seeded into 96- well plates (Costar, #3610) at 4x104 cells/well in antibiotic-free medim.
  • the suspended cells were immediately co-transfected with 60 ng of the Photinus pyralis firefly luciferase reporter plasmid pGL3-control (Promega) and 40 ng of the Renilla reniformis luciferase plasmid pTK-Renilla (a modified version of pGL4.74; Promega) per well using Lipofectamine 2000 as recommended
  • the medium was changed 6 hours post-transfection to include penicillin, streptomycin and doxycycline and the incubation continued for another 18 hours.
  • the activities of the two luciferases were measured 24 hours post- transfection using Dual-Glo Luciferase Assay System (Promega) and a
  • RNA samples were treated with 50 units/ml of RQ1 DNase (Promega) at 37°C for 30 min to remove genomic DNA contamination.
  • Reverse transcription (RT) was carried out using Superscript III (Invitrogen) and random decamer (N10) primers at 50°C for 1 hour.
  • cDNA samples were analyzed by PCR using Taq DNA polymerase (KAPA Biosystems) or quantitative PCR using Fast SYBR Green Master Mix and a StepOnePlus Real-Time PCR System (Applied Biosystems) as recommended. The corresponding primer sequences are listed in the Table 1.
  • RT-PCR products were analyzed by electrophoresis in 2% NuSieve 3:1 (Lonza), 1 ⁇ ⁇ agarose gels.
  • the qPCR reactions were carried out in triplicate and the data were normalized against the HPRT mRNA levels.
  • the colonies were stained with 0.1 % methylene blue in 50% methanol and counted.
  • the titres were calculated as colony forming units (cfu) per milliliter by averaging the colony counts multiplied by the corresponding dilution factors.
  • the titres were normally in the range of 0.5-1 *106 cfu/ml.
  • Genomic DNA isolation [00129]. To isolate genomic DNA, 70% confluent cell cultures grown in 10 cm dishes were trypsinized and pelleted at 500xg for 5 min in 15 ml falcon tubes. The cell pellets were washed with 1.2 ml PBS and lysed in 0.6 ml of DNA extraction buffer containing 100 mM Tris-HCI, pH 7.4, 200 mM NaCI, 5 mM EDTA, 0.2 %SDS and 0.1 mg/ml proteinase K (Fermentas).
  • the lysates were incubated overnight at 55°C followed by subsequent phenol, phenol-chloroform (1 :1 ) and chloroform extractions and genomic DNA precipitation with 0.7 volumes of isopropanol at room temperature. DNA pellets were washed with 70% ethanol and rehydrated in 10 mM Tris-HCI, pH 8.0.
  • Genomic DNAs from the RMCE acceptor cell lines were analyzed by multiplex PCR using Taq DNA polymerase (KAPA Biosystems) and either the 5' (EF, BR, and PR; Table 1 ) or the 3' junction primer mixtures (GF, BF, and WR; Table 1 ).
  • the mixtures were designed to generate distinct PCR products from the acceptor locus before and after inserting the RIPE cassette.
  • the PCR program consisted of a 3 min 95°C step followed by 37 cycles of melting (94°C, 20 sec), annealing (56°C, 30 sec) and elongation (72°C, 90 sec).
  • DMSO was added to the 5' junction reactions to the final concentration of 5% to facilitate the amplification of the GC-rich Pur gene.
  • Control PCR reactions were carried out using primers hGAPDH_F1 and hGAPDH_R1 (Table 1 ) detecting both the bona fide GAPDH gene and a GAPDH pseudogene under the conditions employed.
  • PCR fragment was labeled using a Megaprime DNA labeling System (GE Healthcare) and [ ⁇ -32 ⁇ ]- dCTP (Perkin Elmer) and purified from non-incorporated nucleotides using G-50 spin columns (Geneaid Biotech).
  • Hybridizations were carried out in ExpressHyb hybridization buffer (Cloiitech) as recommended and the radioactive signal was visualized using a Typhoon Trio imager (GE Healthcare).
  • Proteins were extracted from PBS-washed cells with 20 mM Tris-HCI, pH 7.5, 150 mM NaCI, 5 mM EDTA, 10% glycerol, 1 % Nonidet P-40, 1 mM PMSF and 1 ⁇ Complete EDTA-free protease inhibitor cocktail (Roche), separated by 4-20% gradient SDS-PAGE (Bio-Rad), and immunoblotted using the following primary antibodies: mouse monoclonal anti-PTBP1 (ZYMED; 1 :1000 dilution), rabbit monoclonal anti-Ago2 (Cell Signalling Technology; 1 :1000), goat polyclonal anti-TUT4/ZCCHC1 (Imgenex; 1 :500), or mouse monoclonal anti-GAPDH (Ambion; 1 :10,000). The protein bands were visualized using corresponding horseradish peroxidase-conjugated secondary antibodies (mouse and rabbit, GE Healthcare; goat, Santa Cruz Biotechnology) and ECL
  • the invention described herein may include one or more range of values (e.g. size, concentration etc).
  • a range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.

Abstract

Nous décrivons ici une technologie d'échange de cassettes induite par la recombinase (CREM) à haut rendement et faible bruit de fond (HILO), qui produit en quelques jours pour un effort minimal des populations de cellules génétiquement homogènes contenant des éléments d'ARNsh inductibles par la doxicycline. Pour assurer une utilité immédiate de cette plate-forme pour un milieu de la recherche plus étendu, nous avons modifié 11 lignées cellulaires humaines (A549, HT1080, HEK293T, HeLa, HeLa-S3 et U2OS) et murines (CAD, L929, N2a, NIH 3T3 et P19), couramment utilisées, pour les rendre compatibles avec le processus HILO- CREM. En démonstration du principe, nous avons utilisé les lignées cellulaires nouvellement créées pour optimiser le silençage d'une gamme de protéines d'interaction avec l'ARN cellulaire, silençage induit par l'ARNsh. Du fait de sa simplicité technique et de sa rentabilité, la nouvelle plate-forme se révèle très avantageux tant pour les expérimentations à faible rendement que pour les expérimentations à haut rendement faisant intervenir l'ARNsh. Nous avons également apporté la preuve que cette technologie facilite une large gamme d'applications en biologie moléculaire et cellulaire, en ce qu'elle autorise une ingénierie rapide de cellules exprimant essentiellement un quelconque transgène d'intérêt.
PCT/SG2012/000238 2011-07-05 2012-07-05 Nouveau procédé et réactif autorisant des modifications génétiques rapides dans des cellules eucaryotes WO2013006142A1 (fr)

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WO2020200983A1 (fr) 2019-03-29 2020-10-08 F. Hoffmann-La Roche Ag Procédé de génération d'une cellule exprimant fcrn par intégration ciblée de multiples cassettes d'expression dans une organisation définie
WO2020254357A1 (fr) 2019-06-19 2020-12-24 F. Hoffmann-La Roche Ag Procédé de production d'une cellule exprimant une protéine par intégration ciblée à l'aide d'arnm de cre
WO2020254352A1 (fr) 2019-06-19 2020-12-24 F. Hoffmann-La Roche Ag Procédé de génération d'une cellule exprimant un anticorps trivalent par intégration ciblée de multiples cassettes d'expression dans une organisation définie
WO2020254351A1 (fr) 2019-06-19 2020-12-24 F. Hoffmann-La Roche Ag Procédé de génération d'une cellule exprimant un anticorps multivalent, multispécifique par intégration ciblée de multiples cassettes d'expression dans une organisation définie
WO2020254356A1 (fr) 2019-06-19 2020-12-24 F. Hoffmann-La Roche Ag Procédé de génération d'une cellule exprimant un anticorps multivalent, bispécifique par intégration ciblée de multiples cassettes d'expression dans une organisation définie
WO2020254355A1 (fr) 2019-06-19 2020-12-24 F. Hoffmann-La Roche Ag Procédé de génération d'une cellule exprimant un anticorps bivalent bispécifique par intégration ciblée de multiples cassettes d'expression dans une organisation définie

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WO2020084034A1 (fr) 2018-10-26 2020-04-30 F. Hoffmann-La Roche Ag Procédé de criblage d'anticorps multispécifiques utilisant un échange de cassette à médiation par recombinase
WO2020200983A1 (fr) 2019-03-29 2020-10-08 F. Hoffmann-La Roche Ag Procédé de génération d'une cellule exprimant fcrn par intégration ciblée de multiples cassettes d'expression dans une organisation définie
WO2020254357A1 (fr) 2019-06-19 2020-12-24 F. Hoffmann-La Roche Ag Procédé de production d'une cellule exprimant une protéine par intégration ciblée à l'aide d'arnm de cre
WO2020254352A1 (fr) 2019-06-19 2020-12-24 F. Hoffmann-La Roche Ag Procédé de génération d'une cellule exprimant un anticorps trivalent par intégration ciblée de multiples cassettes d'expression dans une organisation définie
WO2020254351A1 (fr) 2019-06-19 2020-12-24 F. Hoffmann-La Roche Ag Procédé de génération d'une cellule exprimant un anticorps multivalent, multispécifique par intégration ciblée de multiples cassettes d'expression dans une organisation définie
WO2020254356A1 (fr) 2019-06-19 2020-12-24 F. Hoffmann-La Roche Ag Procédé de génération d'une cellule exprimant un anticorps multivalent, bispécifique par intégration ciblée de multiples cassettes d'expression dans une organisation définie
WO2020254355A1 (fr) 2019-06-19 2020-12-24 F. Hoffmann-La Roche Ag Procédé de génération d'une cellule exprimant un anticorps bivalent bispécifique par intégration ciblée de multiples cassettes d'expression dans une organisation définie

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