WO2003040318A2 - Procede et systeme de clonage recombinatoire inductible de cellules bacteriennes - Google Patents

Procede et systeme de clonage recombinatoire inductible de cellules bacteriennes Download PDF

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WO2003040318A2
WO2003040318A2 PCT/US2002/035217 US0235217W WO03040318A2 WO 2003040318 A2 WO2003040318 A2 WO 2003040318A2 US 0235217 W US0235217 W US 0235217W WO 03040318 A2 WO03040318 A2 WO 03040318A2
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recombinase
donor
plasmid
receiver
cassette
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PCT/US2002/035217
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WO2003040318A3 (fr
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Patrick Y. Lu
Qingquan Tang
Martin C. Woodle
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Intradigm Corporation
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Publication of WO2003040318A3 publication Critical patent/WO2003040318A3/fr

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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
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host

Definitions

  • the field of the present invention is a method and system for the high throughput screening of genes or DNA sequences with expressed sequence tags using in vivo recombinational cloning. Sequences cloned are inserted into shuttle vectors by the in vivo induction of recombinase creating a target vector optimized for mammalian expression.
  • Genomics is the study of the DNA sequences, the identification of genes and the analysis of the physiological function of those genes.
  • the knowledge derived from genomics will allow for an understanding of the molecular processes that underlie life and also revolutionize agriculture, medicine, and many aspects of healthcare.
  • human gene discovery and functional genome studies have been dramatically enhanced by the Human Genome Project, and by tremendous effort from the entire scientific community and biomedical industry. As the Human Genome Project nears completion and the sequences of over 30,000 genes are identified the next goal is to identify their function and analyze their effects in vivo.
  • full-length cDNA Another advantage of the use of full-length cDNA is that with it gene delivery can be adapted into a high- throughput process for the rapid investigation of the physiologic actions of gene products.
  • a more efficient gene delivery technology is needed in the post-genome era and will be a powerful tool for functional genomic study.
  • the fast growing pools of full-length cDNA and nucleic acid sequences bearing expressed sequence tags require better and more sophisticated gene delivery vehicles.
  • the invention provides methods and systems for the inducible recombinational cloning of target sequences in bacterial cells, and also methods and systems for the expression and analysis of transfection efficiency in mammalian host cells or organisms.
  • the invention also provides an improved means of the subcloning of the target gene or PCR product into the vector greatly facilitating that procedure.
  • One embodiment of the invention is directed to a cloning method and system wherein a transgene (e.g. a target gene or PCR product), is transferred into a donor vector via a topoisomerase cut-and-joining function, and is translocated into a receiver plasmid, through the action of recombinase, to create a target vector which is specifically designed to be an efficient mammalian expression vector.
  • a transgene e.g. a target gene or PCR product
  • the donor plasmid may contain a bacterial origin of replication and a selectable marker comprising antibiotic resistance gene (ARG).
  • the donor plasmid may contain a donor cassette that comprises a gene or nucleic acid sequence bearing an expressed sequence tag and further comprises a splicing acceptor site, a topoisomerase site and/or a multiple cloning site (MCS), a polyadenylation (poly A) site and a selectable marker conferring a different antibiotic resistance from that found in the backbone of the donor plasmid.
  • MCS multiple cloning site
  • poly A polyadenylation
  • the 5' and 3' ends of the donor cassette are marked by recombinase recognition motifs.
  • the transgene or PCR product can be easily ligated into the donor plasmid at the site of the donor cassette, either by the use of topoisomerase or through the use of a conventional multiple cloning site (MCS).
  • MCS multiple cloning site
  • Another embodiment of the invention that is directed to a receiver plasmid that contains a selectable marker in the backbone, that may be the same or different as that found in the backbone of the donor plasmid.
  • the receiver plasmid contains a recombinase cassette which comprises recombinase recognition motif compatible with the recombinase recognition motifs found in the donor plasmid.
  • the recombinase cassette also contains a recombinase gene which is under the control of an inducible promoter such that induction of the gene results in the production of the recombinase enzyme.
  • the recombinase cassette comprises an ARG, in the opposite orientation to the recombinase gene and different from that found in the backbone of the receiver plasmid and donor plasmid or donor cassette.
  • 5' to the recombinase cassette is a viral promoter, expressed in eukaryotic cells, and an intron.
  • 3" to the recombinase cassette is a further inducible promoter in the same orientation as the antibiotic resistance marker gene found in the recombinase cassette.
  • the donor cassette By cotransfection of the host cells with the donor and receiver plasmids followed by induction of the recombinase gene in the recombinase cassette the donor cassette is translocated to the receiver plasmid.
  • the recombinase cassette lacking an origin of replication is not replicated in the bacterial cells and is, therefore not propagated.
  • the translocation of the donor cassette to the receiver plasmid creates a target vector or plasmid which now possesses a new combination of selectable markers derived from the backbone of the receiver plasmid and the donor cassette of the donor plasmid.
  • the target vector can then be amplified in the bacterial host cells and is maintained through selection using the new combination of antibiotic resistance that results from the translocation. Because of this selection process the target vector can be maintained in the host cells and a library of genes or PCR products may be maintained in bacterial cultures.
  • the target vector is designed to incoiporate viral promoters, splice sites, intron sequences and mammalian expression sequences it is an efficient expression vector for use to directly transfect mammalian cells or tissues.
  • the arrangement of the splice donor, intron, recombinase recognition motif and splice acceptor site allows those sequences to be spliced out upon transcription resulting in an mRNA sequence in which the viral promoter is directly followed the transgene of interest and a polyadenylation sequence allowing for extremely efficient translation of the target gene.
  • This arrangement provides the means to study the function of the gene in mammalian cells, with very little manual manipulation of the DNA or vectors, by use of the host cells machinery to perfonn the molecular handling of the DNA and amplify the resulting plasmids.
  • Another embodiment of the invention is directed to bacterial cells that are transfected with a transformer plasmid comprising a bacterial origin of replication, an antibiotic resistance marker gene and an inducible promoter followed by a recombinase gene.
  • the donor plasmid is the same as described previously while, the receiver plasmid contains a recombinase cassette but lacks the recombinase gene.
  • the receiver plasmid contains a recombinase cassette but lacks the recombinase gene.
  • the donor cassette is translocated to the receiver plasmid, conferring a new combination of antibiotic resistance marker genes.
  • FIG. 1 Schematic Diagram of the Donor Plasmid.
  • Figure 4 A schematic diagram representing the cottansfection of bacteria with the Donor and Receiver plasmids.
  • Figure 5 The recombination that occurs in bacterial cells.
  • Figure 8 Illustrates the in vivo generation of shuttle and expression vectors using various viral promoters.
  • Figure 9. Illustrates the insertion of the donor plasmid into the different forms of the receiver plasmid shown in figure 8.
  • Figure 10 Illustrates the stable transfection of a mammalian 293 cell with a target vector having an adenoviral promoter.
  • FIG. 11 This figure illustrates that upon transfection of mammalian cells with the target vector, the post-transcriptional process results in the splicing out of the intron, LoxP splice acceptor sequence, resulting in an mRNA product.
  • Figure 14 Another example of successful transfer of more donor cassettes into the receiver plasmids.
  • the present invention overcomes the problems and disadvantages associated with cuiTent sttategies and designs and provides new and improved methods for the study of genomics by allowing the efficient interpretation of the functional properties of human genes.
  • the method provides surprisingly more efficient mechanisms to generate complete sets of full-length cDNA clones and sequences for human and model-organism genes.
  • the disclosed method allows a greater ability to study gene expression and control and creation of mutations that cause loss of function in non-human organisms.
  • the invention also provides new methods for the in vivo inducible recombinational cloning in bacterial host cells.
  • the invention allows the recombination of a donor cassette found in a donor plasmid to a receiver plasmid by site specific translocation in vivo such that the subcloning from a shuttle vector, appropriate for bacterial amplification, to an expression vector appropriate for eukaryotic expression is carried out completely within the host cell.
  • the resulting target vector is optimized to provide important sequences to facilitate the tianscription of the transgene contained in the donor cassette.
  • the instant invention also allows for ease of cloning of the gene or PCR product directly into the donor plasmid through the use of a topoisomerase action motif.
  • a "lox site” refers to a nucleotide sequence at which the product of the ere gene of bacteriophage PI, Cre recombinase, can catalyze a site-specific recombination.
  • a variety of lox sites are known to the art including the naturally occurring loxP (the sequence found in the PI genome), loxB, loxL and loxR (these are found in the E. coli chromosome) as well as a number of mutant or variant lox sites such as loxP511, lox.DELTA.86, lox.DELTA.l 17, loxC2, loxP2, loxP3 and loxP23.
  • a recombinase refers to enzymes that recognize and bind to a short nucleic acid site or sequence and catalyze the recombination of nucleic acid in relation to these sites.
  • a "recombinase recognition motif” refers to a short nucleic acid site or sequence which is recognized by recombinase and which become the crossover regions during the site-specific recombination event.
  • Examples of recombinase recognition motifs include, but are not limited to, lox sites, fit sites, art sites and dif sites.
  • the term "frt site” refers to a nucleotide sequence at which the product of the FLP gene of the yeast 2 .mu.m plasmid, FLP recombinase, can catalyze a site- specific recombination.
  • the present invention provides methods and a system for the inducible recombinational cloning of a donor cassette, bearing a transgene for functional genomic study, with a receiver plasmid using site specific DNA recombinase (Cre) in bacterial cells and includes construction of several key components into the plasmids to create efficient mammalian expression vectors.
  • the methods described in the present invention are specifically useful for HTP generation of expression vectors (viral or non-viral) in mammalian cell systems and, as a result, provide a significant improvement over current technologies.
  • expression vectors refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism.
  • Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences.
  • Eukaryotic cells are known to utilize promoters, enhancers, Kozak sequences, and termination and polyadenylation signals. By use of these genetic components foreign DNA may be more efficiently expressed in model systems.
  • transformation and “transfection” refer to the introduction of foreign DNA into prokaryotic or eukaryotic cells. Transformation of prokaryotic cells may be accomplished by a variety of means known to the art including the treatment of host cells with CaCl 2 , and electroporation, etc. Transfection of eukaryotic cells may be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.
  • a donor plasmid denotes a series of nucleotide sequences bearing a bacterial origin of replication and a selectable marker such that it can be specifically propagated in vivo.
  • the term donor cassette denotes a series of nucleotide sequences flanked by recombinase recognition motifs which are capable of being translocated in a site specific manner from the site of one recognition motif to another.
  • the invention provides a donor plasmid that comprises a donor cassette comprising a transgene for study and flanked by recombinase recognition motifs or lox sites.
  • the invention also comprises a receiver plasmid that contains a transgene cassette, which comprises a recombinase gene under the control of an inducible promoter and is also flanked by lox sites compatible with those of the donor cassette.
  • a transgene cassette which comprises a recombinase gene under the control of an inducible promoter and is also flanked by lox sites compatible with those of the donor cassette.
  • the cassette includes sequences important for the optimum translation in mammalian cells.
  • Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and acceptor site (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989) pp. 16.7-16.8).
  • a commonly used splice donor and acceptor site is the splice junction from the 16S RNA of S ⁇ O.
  • these sequences may include but are not limited to Kozak sequences and polyadenylation sequences.
  • the Kozak sequence is based on upon the published consensus sequence for the translation start site consensus sequence of mRNAs, in some instances the Kozak consensus sequence is (G/A)NNATGG, (Kozak, 1991, Jour. Cell Biology, Vol. 115, pp. 887-903) (hereby incorporated in its entirety).
  • the polyadenylation site denotes a DNA sequence, which directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly A tail are unstable and are rapidly degraded.
  • the poly A signal utilized in an expression vector may be "heterologous" or "endogenous.”
  • An endogenous poly A signal is one that is found naturally at the 3' end of the coding region of a given gene in the genome.
  • a heterologous poly A signal is one which is isolated from one gene and placed 3' of another gene.
  • the cassette has a selectable marker comprising an antibiotic resistance marker gene such that the donor cassette can be specifically selected for.
  • the donor cassette it comprises a Topoisomerase I Action Motif and/or a multiple cloning site so that the transgene of interest can be ligated directly into the donor cassette within the donor plasmid.
  • the backbone of the donor plasmid also contains a bacterial origin of replication and second selectable marker which is different from the selectable marker found within the donor cassette. This combination allows the plasmid to be propagated in bacteria and ensures that the plasmid is maintained in E. coli or another appropriate bacterial strain with the due to the selectable marker found in the donor cassette. It is a further aspect of the invention that the target transgene or PCR product may be cloned into the donor plasmid and the plasmid with transgene insert may be maintained in bacterial culture such that a library of cloned donor plasmids may be created and maintained awaiting recombination with the receiver plasmid.
  • the invention also comprises a receiver plasmid that also includes a selectable marker and an origin of replication such that the receiver plasmid may be maintained in bacterial cells.
  • the receiver plasmid also contains a transgene cassette.
  • the recombinase cassette comprises an inducible promoter which drives a recombinase gene which may be the Cre recombinase or the FLP recombinase gene flanked by recombination recognition motifs.
  • a recombinase is an enzyme that recognizes and binds to a short nucleic acid site or sequence and catalyzes the recombination of nucleic acid in relation to these sites.
  • Cre is a 38- kDa site-specific DNA recombinase from bacteriophage PI that mediates recombination between DNA sequences at specific loci, called lox sites. These sites comprise two 13-bp inverted repeats separated by an 8-bp spacer region that provides directionality to the recombination reaction; the fixed directionality of this 8-bp region keeps the orientation and reading frame of the gene of interest intact following transfer to another vector.
  • the recombinase cassette includes another selectable marker gene in the opposite orientation, which is different than that found in the donor cassette or the backbones of either the donor or receiver plasmid.
  • the receiver plasmid contains a strong viral promoter and an intron sequence outside of the recombinase cassette on the 5' side.
  • an inducible promoter On the 3' side of the cassette is an inducible promoter in the same orientation as the antibiotic resistance marker gene found inside the cassette.
  • 3' to the inducible promoter is another viral promoter which drives a reporter gene.
  • the viral promoters may be, but are not limited to, the cytomegalovirus promoter, the rous sarcoma virus promoter the lentivirus promoter, the herpes simplex virus promoter the adenovirus promoter or any other similar promoter.
  • the inducible bacterial promoter may be, but is not limited to the T7/Lac, Tet, trc or Lac promoters.
  • An inducible promoter is a promoter that will turn on transcription of the gene it controls upon stimulation with a particular agent or condition such as change in temperature, addition of IPTG, and many other factors. For example, IPTG: Isopropyl-beta-D-thiogalactoside, induces the lac promoter.
  • IPTG Isopropyl-beta-D-thiogalactoside
  • the architecture of the receiver plasmid allows the inducible bacterial promoter on the 3' side of the recombinase cassette to drive the antibiotic resistance gene within the cassette only after recombinase mediated translocation has occurred. Further, the viral promoter drives the reporter gene only when the plasmid has been transfected to eukaryotic cells.
  • the invention thus allows for bacterial cells to be cotransfected with the donor plasmid and the receiver plasmid each bearing a selectable marker in the plasmid backbone.
  • the selectable markers in the backbones of the plasmids may be the same. It is also evident that the selectable markers may be different such that upon cotransfection the host cells will be resistant to two different antibiotics conferred by the selectable markers in the backbone of the plasmids. Induction of the recombinase gene initiates the translocation of the donor cassette into the receiver plasmid at the site occupied by the recombinase cassette, this construct now forms the target plasmid.
  • the recombinase cassette is removed and since it lacks a bacterial origin of replication it is not propagated in the host cell. Because the donor cassette has a selectable marker different from that of the backbone selectable marker a new combination of selective pressure can be applied, combining the antibiotic resistance encoded by the receiver with that encoded by the donor cassette. This allows only those products which have the correct recombination between receiver plasmid and donor cassette to be selected for and propagated within the host cell.
  • the host cells may be stably transfected with a transformer plasmid which comprises an inducible promoter driving a recombinase gene followed by an antibiotic resistance marker gene in the opposite orientation driven by an inducible ac promoter.
  • the receiver plasmid contains a recombinase cassette flanked by recombinase recognition motifs, as described above, but it lacks the recombinase gene.
  • the cells are cotransfected with the receiver plasmid and the donor plasmid and the induction of the recombinase gene allows the translocation of the donor cassette to the receiver cassette creating the target plasmid and conferring a new combination of antibiotic resistance as previously described.
  • the formation of the target vector brings the active sequences of the donor cassette into functional alignment with the genes that previously flanked the recombinase cassette of the receiver plasmid.
  • the target vector represents a combination of genes thus creating a mammalian expression vector.
  • This vector comprises in 5' to 3' order a viral promoter, suitable for strong constitutive expression in mammalian cells, such as the cytomegalovirus promoter, an intron sequence, a recombinase recognition motif, a splicing acceptor site, a possible Kozak sequence, and the transgene of investigation, a polyadenylation sequence, all of these will facilitate gene expression in mammalian cells.
  • the resulting vector also possesses the antibiotic resistance gene that is in the opposite (3 '-5') orientation from the rest of the nucleotide sequences received from the donor plasmid.
  • these sequences are followed by the second recombinase recognition motif, the latter is followed by a second viral promoter such as the rous sarcoma virus promoter.
  • the viral promoter drives a reporter gene which in some cases may be the green fluorescent protein or any other gene product that according to the researcher's choice. Because the reporter gene is driven by a viral promoter it is silent until the target plasmid is transfected into mammalian cells. Once the target plasmid is created and selected for by the appropriate combination of antibiotics, the vector can then be purified from the E.
  • the construct is uniquely suited for expression in mammalian cells.
  • the intron sequence will be spliced out between the splice donor site and the splice acceptor site . This process also removes the recombinase recognition motif situated between the two splice sites.
  • the recombination, cloning and selective steps all occur in vivo, hence there is no need for any of the apparatus currently necessary for in vitro cloning, including varying buffer solutions for each step gel purification and serial amplification of the target gene sequence. These steps all occur in the bacterial host cells.
  • the selectable markers may include antibiotic resistance genes or genes which encode an enzymatic activity that confers the ability to grow in medium lacking what would otherwise be an essential nutrient.
  • the donor plasmid shown in Figure 1 contains an origin of replication and an antibiotic resistance marker gene in the vector backbone.
  • the donor cassette is illustrated flanked by recombinase recognition motifs.
  • the cassette also contains multiple cloning sites and/or a Topoisomerase I Action Motif (TAM) for insertion of the transgene sequence.
  • TAM Topoisomerase I Action Motif
  • the optional Kozak sequence is also shown in the figure. Since the TAM has the 5'-CCCTT-3' Topo-I recognition sequence, it could be designed to enable the complementing DNA strain to probably position a nucleotide "G" in the "-3) position relative the the start codon ATG, creating a Kozak sequence which will facilitate the expression of mterestd gene in eukaryotic cells.
  • a second antibiotic resistance gene without a promoter sequence is harbored downstream of the MCS in an opposite orientation.
  • a non-translational DNA leader sequence is built in and located between left recmobinase motif and the transgene insertion site.
  • a splice acceptor sequence follows after the left recombinase motif.
  • the Poly A signal sequence follows the insertion site.
  • FIG. 2 Two embodiments of the receiver plasmid are shown in Figure 2.
  • the upper figure shows the presence of the inducible promoter followed by the recombinase gene; the lower figure lacks the promoter and gene.
  • the receiver plasmid contains both an origin of replication and an ARG in the backbone. Both embodiments contain a constitutive ly expressed reporter gene, which indicates transfection efficiency in mammalian cells.
  • Embodiment I A cytomegalovirus (CMV) promoter drives transgene expression in mammalian cells.
  • An intron precedes the recombinase cassette.
  • the recombinase motif is followed by a T7/Lac promoter, which drives the recombinase gene.
  • a kanamycin resistance gene in the opposite orientation, is followed by the second recombinase recognition motif.
  • a second Lac promoter in the same orientation as the kanamycin gene driving expression of that ARG.
  • a second viral promoter follows driving the expression of the reporter gene, in this instance the sequence for the green fluorescent protein.
  • Embodiment II of the invention of the receiver plasmid lacks the promoter/recombinase sequences within the recombinase cassette and there is only one recombinase recognition motif since the design requires insertion of the transgene cassette not translocation of the recombinase gene.
  • Figure 3 shows bacteria permanently transfected with the recombinase gene when the receiver plasmid of Embodiment II.
  • Figure 4 depicts a schematic diagram representing the cotransfection of bacteria with the Donor and Receiver plasmids.
  • Figure 5 illustrates the recombination that occurs in bacterial cells. Upon induction of the recombinase gene the recombinase cassette is spliced out and the donor cassette is inserted. This recombination creates the target vector.
  • Figure 6 shows a schematic diagram illustrating the cotransfection of the Donor Plasmid and the Receiver Plasmid of Embodiment II. This figure illustrates that when Embodiment II of the invention is used the plasmids are transfected into bacterial cells which already are permanently transfected with the recombinase transformation cassette.
  • Figure 7 shows a diagram illustrating the results of the translocation that occurs when embodiment II of the invention.
  • Figure 8 illustrates the in vivo generation of shuttle and expression vectors using various viral promoters.
  • the receiver vector is designed in various forms to fit the purposes of the shuttle vectors for different expression vectors, such as naked plasmid vector and viral vector for direct gene delivery.
  • the Lenti shuttle vector uses the Lenti virus promoter; HSVl, uses the He ⁇ es Simplex I viral promoter; Avl uses the adenovirus promoter and pCMV, uses the cytomegalovirus promoter.
  • Figure 9 illustrates the insertion of the donor plasmid into the different forms of the receiver plasmid shown in figure 8.
  • Figure 10 illustrates the stable transfection of a mammalian 293 cell with a target vector having an adenoviral promoter.
  • Figure 11 illustrates that upon transfection of mammalian cells with the target vector, the post-transcriptional process results in the splicing out of the intron, LoxP splice acceptor sequence, resulting in an mRNA product.
  • Figure 12 shows the expression of IPTG-inducible Cre from the Cre- expressing plasmid.
  • the plasmid expressing IPTG-inducible Cre was constructed, named pIntQE60Cre.
  • the figure shows the IPTG-induction of Cre in two of the pIntQE60-transformed M15(Rep4) bacteria colonies. A mini-induction was carried out, with 2 ml LB, 1 mm IPTG, 2 hours of induction at 37°C. Table 1 provides an in vivo recombination.
  • Figure 13 shows an example of successful transfer of donor cassettes into the receiver plasmids through co-transfection of the cre-expression bacterium.
  • SEAP/Cm chloramhinicol resistance gene
  • LacZ/Cm lacZ/Cm were transferred, respectivele, from donor plasmid into the receiver plasmid, pint-Avl-SpaT.
  • the resulting target plasmid DNAs were isolated from transformant bacteria, and analyzed by Bglll/Xho restriction digestion.
  • Lane 1 The receiver plasmid control, resulting in three DNA fragments (4,2, 2.3, and 0.2 kb).
  • Lane 2 & 3 the receiver with SEAP/Cm insert; because of the insertion, the 4.2 Kb frament changed to 6.6 Kb, as expected.
  • Lane 4 & 5 the receiver with LacZ/Cm insert, the 4.2Kb fragment changed to 8.0 Kb.
  • Figure 14 shows another example of successful transfer of more donor cassettes into the receiver plasmids . Seventeen more cDNAs were transferred into receiver and analyzed with EcoRI digestion the similar way as described in Figure 13. Transfer of four representative genes are presented in this picture. Lane 10 & 11: the receiver alone (Lane 1-3, and 13, IFN- gene-containing receiver. Lane 4-6: TNF- ⁇ . Lane 7-9: IFN- ⁇ . Lane 14-16: VEG-1. Lane 17-19: IL-10. Except lane 6, all the mentioned lanes have shown the reasonable two DNA fragments with reasonable size, indicating successful gene transfer. The upper band of 5.568Kb of the vector had gained extra size of these genes (TNF- ⁇ , 882bp; IFN- ⁇ , 858bp;NEG-l, 1046bp; and IL-10, 854bp).

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Abstract

La présente invention concerne des procédés et des systèmes permettant un criblage à haut débit de gènes et de séquences d'ADN portant des marqueurs de séquence exprimés dans des cellules mammaliennes. L'invention peut facilement être appliquée à la délivrance d'autres fonctions telles que celles d'ARNi, de ribozyme, d'antisens, de vaccin à ADN, etc. L'invention comprend un plasmide donneur portant une cassette de recombinase qui peut se propager ou se conserver dans une culture bactérienne. L'utilisation du clonage recombinatoire inductible permet de recombiner facilement et efficacement in vivo la cassette du donneur qui comprend une séquence transgénique et un plasmide receveur, afin de créer un vecteur cible comprenant des motifs désirés (par exemple, des sites d'épissage, des introns et des séquences de polyadénylation). L'invention se rapporte également à des moyens pratiques permettant d'introduire le transgène dans la cassette du donneur et elle permet de créer une bibliothèque de séquences transgéniques destinées à l'étude qui sont conservées soit dans le plasmide donneur soit dans le vecteur cible. Etant donné la facilité avec laquelle les gènes et gènes putatifs peuvent être introduits dans le plasmide donneur, et le fait que cette recombinaison s'effectue in vivo, l'invention permet une étude fonctionnelle de la génomique dans des systèmes modèles.
PCT/US2002/035217 2001-11-02 2002-11-04 Procede et systeme de clonage recombinatoire inductible de cellules bacteriennes WO2003040318A2 (fr)

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