WO2002004621A2 - Methodes permettant de creer des constructions utilisees pour introduire des sequences dans des cellules souches embryonnaires - Google Patents

Methodes permettant de creer des constructions utilisees pour introduire des sequences dans des cellules souches embryonnaires Download PDF

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WO2002004621A2
WO2002004621A2 PCT/US2000/018812 US0018812W WO0204621A2 WO 2002004621 A2 WO2002004621 A2 WO 2002004621A2 US 0018812 W US0018812 W US 0018812W WO 0204621 A2 WO0204621 A2 WO 0204621A2
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construct
sequence
sequences
dna
vector
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PCT/US2000/018812
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WO2002004621A3 (en
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Robert D. Klein
Thomas J. Brennan
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Deltagen, Inc.
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Priority to PCT/US2000/018812 priority Critical patent/WO2002004621A2/fr
Priority to AU2000260840A priority patent/AU2000260840A1/en
Publication of WO2002004621A2 publication Critical patent/WO2002004621A2/fr
Publication of WO2002004621A3 publication Critical patent/WO2002004621A3/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • 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
    • 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
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • 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
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • 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
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • This invention is in the field of molecular biology and medicine. More specifically, it relates to novel vector constructs and to methods of making DNA constructs for introducing targeted mutations into embryonic stem cells.
  • ESTs expressed sequence tags
  • conventional targeting DNA vectors contain (1) two blocks of DNA sequences that are homologous to separate regions ofthe target site; (2) a DNA sequence that codes for resistance to the compound G418 (Neo 1 ) between the two blocks of homologous DNA (i.e. positive selection marker) and (3) DNA sequences coding for herpes simplex virus thymidine kinases (HSN-tkl and HSV-tk2) outside ofthe homologous blocks (i.e. negative selection marker).
  • HSN-tkl and HSV-tk2 herpes simplex virus thymidine kinases
  • the present invention provides novel constructs (e.g., plasmid vectors) useful in a rapid and efficient method for generating DNA constructs suitable for introduction into embryonic stem cells.
  • novel methods described herein eliminate the need for the traditional hybridization isolation of a single genomic clone, restriction mapping ofthe clone and multiple cloning steps.
  • the present invention provides an unexpected reduction in the time required for making a "knock-out" vector. Methods described in the art require 2 to 4 months to accomplish what the claimed invention can achieve within 1-2 weeks.
  • the unexpected increase in efficiency accomplished by the methods described herein involves methods that have not previously been applied to the process of making a "knock-out" vector, including identification of a complex mixture containing the clone of interest, long-range polymerase chain reaction (PCR) and ligation independent cloning.
  • the present inventors are the first to generate a construct without isolating an individual genomic clone or mapping the restriction sites within the clone.
  • the inventors are also the first to generate knock-out constructs using ligation independent cloning, including four- way annealing of nucleotide fragments.
  • the subject invention provides novel constructs and efficient methods of making constructs which, when introduced into embryonic stem cells, deletes or mutates a specific gene in the target animal.
  • the invention includes a nucleotide construct comprising a sequence encoding a positive selection marker flanked by restriction enzyme sites.
  • the restriction enzyme sites are flanked, on the side opposite the positive selection marker, by sequences which are not complementary to each other and which do not include one ofthe four types of base pairs at any position.
  • the vector construct can be treated so that single-stranded regions are created at each sequence flanking one side ofthe restriction enzyme sites.
  • the nucleotide construct comprises a sequence encoding a positive selection marker flanked on each side by at least one restriction enzyme site.
  • the restriction enzyme site on each side ofthe positive selection marker is a unique site.
  • Each ofthe aforementioned restriction enzyme site is flanked by a pair of annealing sites which do not contain at least one type of base at any position.
  • the construct can be treated to create single-stranded regions and this creates the pair of annealing sites. None ofthe four annealing sites are complementary to each other so that when single-stranded regions are created, they cannot anneal to each other to reseal the vector, i.e., the single stranded regions are incompatible overhangs. However, the single stranded overhangs are compatible with, and can anneal to, the single stranded ends of insert fragments containing sequences homologous to the target gene or a target sequence.
  • the restriction enzyme sites and annealing sites are designed for directional cloning.
  • Plasmid pDG2 contains a unique restriction site, Sac II, between annealing sites 1 and 2 flanking one side ofthe positive selection marker (Neo r in this case), and another unique restriction site, Sac I lying between annealing site 3 and site 4 flanking the other side ofthe positive selection marker.
  • single-stranded regions are created by treating the vector with the appropriate restriction enzymes and with a DNA polymerase, for instance, T4 DNA polymerase.
  • a DNA polymerase for instance, T4 DNA polymerase.
  • the construct comprises a plasmid vector and the positive selection marker is a neomycin resistance gene (Neo 1 ).
  • the screening marker on the side ofthe restriction enzyme sites outside the regions ofthe construct which are homologous to the target sequence, shown for example in Figure 7, as opposite the positive selection marker.
  • the screening marker can be green fluorescent protein (GFP) or a modified fluorescent protein.
  • the construct ofthe present invention also includes a negative selection marker on the side ofthe restriction sites opposite the positive selection marker (e.g., next to the plasmid backbone sequences).
  • the negative selection marker can be thymidine kinase (tk).
  • tk thymidine kinase
  • the construct is the plasmid vector "pDG2" and has the sequence shown in SEQ ID NO:l .
  • the construct can also be the plasmid vector "pDG4,” as shown in SEQ ID NO:2.
  • the invention provides a method of making a DNA construct useful in introducing a nucleotide sequence into a target DNA, comprising (a) amplifying a polynucleotide comprising two different nucleotide sequences substantially homologous to the target DNA; and (b) inserting a gene encoding for a positive selection marker between the two different nucleotide sequences substantially homologous to the target DNA.
  • the positive selection marker may be, for example, a neomycin resistance gene (Neo 1 ).
  • the amplification step is performed in one-step from a genomic DNA library using, for example, oligonucleotide primers in a PCR reaction.
  • the library is a plasmid library.
  • the amplified polynucleotide further comprises a gene encoding a selectable marker, for example, a gene encoding for ampicillin resistance.
  • the vector can also include a second sequence coding for a screening marker, for example, green fluorescent protein (GFP), or another modified fluorescent protein.
  • GFP green fluorescent protein
  • the present invention also includes a method of making a DNA construct useful in introducing a nucleotide sequence into a target DNA, comprising: (a) providing a polynucleotide(s) substantially homologous to the target DNA; (b) generating two different fragments ofthe polynucleotide(s); (c) providing a vector having a gene encoding for a positive selection marker; and (d) using ligation independent clomng to insert the two different fragments into the vector to form the construct, wherein the positive selection marker is between the two different sequence fragments in the construct.
  • the positive selection marker can be a neomycin resistance gene (Neo 1 ) and the vector may be pDG2 (SEQ ID NO:l) or pDG4 (SEQ ID NO:2).
  • the vector can also include a second sequence coding for a screening marker, for example, green fluorescent protein (GFP) or another modified fluorescent protein.
  • the vector can also include a second sequence coding for a negative selection marker.
  • the method includes PCR amplifying the fragments with oligonucleotide primers having 5' sequences which do not have one ofthe four base pairs at any position (also referred to herein as lacking one nucleotide).
  • the 5' sequences lacking one type of base are at least 5, preferably 12, even more preferably at least 20 to 25 nucleotides in length.
  • the oligonucleotide sequences are shown in
  • the present invention also includes a method of making a DNA construct wherein the ligation independent cloning is performed in one step or in two steps.
  • the invention also provides a method of disrupting the function of a target sequence or gene in a cell by (a) inserting sequences homologous to the target gene into a construct ofthe invention as described above, such that the sequences homologous to the target gene flank the positive selection marker, to produce a targeting construct; and (b) introducing the targeting construct into the cell to produce a homologous recombinant wherein the function ofthe target gene or sequence is disrupted.
  • the cell is an ES cell.
  • a targeting construct produced by this method is also provided.
  • Another aspect ofthe invention is a method of enriching for the desired non- random integrant ofthe targeting vector wherein homologous recombination between the targeting vector and the target sequence or gene has mutated or disrupted the target gene.
  • the enrichment step involves screening cells that have taken up the targeting construct, with ultraviolet light and identifying cells that do not fluoresce, for further testing by PCR or other methods to confirm the targeted mutation.
  • the invention includes a host cell or an animal containing a construct described herein.
  • the construct is a targeting construct, preferably, the targeting construct disrupts the function ofthe target gene within the host cell or animal.
  • Figure 1 is a schematic depicting one method of constructing a targeting vector of the present invention.
  • the plasmid PCR method is described in Examples 9 and 10.
  • Figure 2A is a schematic depicting the pDG2 vector.
  • the vector contains an ampicillin resistance gene and a neomycin (Neo 1 ) gene. On each side ofthe Neo r gene are two sites for ligation independent cloning along with restriction sites.
  • the sequence of pDG2 is shown in Figure 2B and SEQ ID NO:l.
  • Figure 3A is schematic depicting the pDG4 vector.
  • the vector contains an ampicillin resistance gene, a neomycin (Neo 1 ) gene and a green fluorescent protein (GFP) gene.
  • GFP green fluorescent protein
  • On each side ofthe Neo r gene are two sites for ligation independent cloning along with restriction enzyme recognition sites.
  • the sequence of pDG4 is shown in Figure 3B and SEQ ID NO:2.
  • Figure 4 shows the nucleic acid sequence before and after T4 polymerase treatment of annealing sites 1-4 contained on the ends of PCR amplified genomic DNA.
  • Figure 5 shows the nucleic acid sequence before and after T4 polymerase treatment of annealing sites 1-4 contained within the pDG2 vector.
  • Figure 6 shows the arrangement of 5' and 3' flanking DNA relative to annealing sites 1 , 2, 3 and 4 within the pDG2 vector during an annealing reaction.
  • Figure 7 shows the arrangement of 5' and 3' flanking DNA relative to annealing sites 1 , 2, 3 and 4 and the GFP screening marker within the pDG4 vector during an annealing reaction.
  • Figure 8 shows the sequences ofthe oligonucleotide primers (SEQ ID NO: 19 through SEQ ID NO:44) used in Examples 4 to 10.
  • the lower case sequences are to cloning sites (e.g. ligation independent cloning sequences).
  • the present invention provides a novel fast and efficient method of making a construct suitable for introducing targeted mutations into embryonic stem (ES) cells.
  • the construct is generated in two steps by (1) amplifying (for example, using long-range PCR) sequences homologous to the target sequence, and (2) inserting another polynucleotide (for example a selectable marker) into the PCR product so that it is flanked by the homologous sequences.
  • the vector is a plasmid from a plasmid genomic library.
  • the completed construct is also typically a circular plasmid.
  • using long-range PCR with "outwardly pointing" oligonucleotides results in a vector into which a selectable marker can easily be inserted, preferably by ligation independent cloning.
  • the construct can then be introduced into ES cells, where it can disrupt the function ofthe homologous target sequence.
  • two separate fragments of a clone of interest are amplified and inserted into a vector containing a positive selection marker using ligation independent cloning techniques.
  • the clone of interest is generally from a phage library and is identified and isolated using PCR techniques.
  • the ligation independent cloning can be performed in two steps or in a single step. The methods ofthe present invention typically result in a finished construct within one week and is thus much more rapid than the several months currently needed to make a knock-out construct using conventional techniques.
  • polynucleotide and “nucleic acid molecule” are used interchangeably to refer to polymeric forms of nucleotides of any length.
  • the polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their analogs. Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotide includes single-, double-stranded and triple helical molecules.
  • Olionucleotide refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double-stranded DNA.
  • Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art.
  • a "primer” refers to an oligonucleotide, usually single-stranded, that provides a 3'-hydroxyl end for the initiation of enzyme-mediated nucleic acid synthesis.
  • polynucleotides a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a nucleic acid molecule may also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs.
  • Analogs of purines and pyrimidines are known in the art, and include, but are not limited to, aziridinycytosine, 4-acetylcytosine, 5- fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl- aminomethyluracil, inosine, N6-isopentenyladenine, 1 -methyl adenine, 1- methylpseudouracil, 1 - ethylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3 -methyl cytosine, 5-methylcytosine, pseudouracil, 5- pentylnyluracil and 2,6-diaminopurine.
  • the use of uracil as a substitute for thymine in a deoxyribonucleic acid is also considered an analogous form of pyrimidine.
  • a “fragment” (also called a "region") of a polynucleotide is a polynucleotide comprised of at least 9 contiguous nucleotides, preferably at least 15 contiguous nucleotides and more preferably at least 45 nucleotides, of coding or non-coding sequences.
  • base pair also designated “bp,” refers to the complementary nucleic acid molecules.
  • purine adenine (A) is hydrogen bonded with the pyrimidine base thymine (T), and the purine guanine (G) with pyrimidine cytosine (C).
  • Each hydrogen bonded base pair set is also known as Watson- Crick base-pairing.
  • a thousand base pairs is often called a kilobase pair, or kb.
  • a “base pair mismatch” refers to a location in a nucleic acid molecule in which the bases are not complementary Watson-Crick pairs.
  • nucleotide sequence which does not have one ofthe four bases at any position.
  • a sequence lacking one nucleotide i.e., lacking one type of base
  • construct refers to an artificially assembled DNA segment to be transferred into a target tissue, cell line or animal, including human.
  • the construct will include the gene or a sequence of particular interest, a marker gene and appropriate control sequences.
  • plasmid refers to an autonomous, self- replicating extrachromosomal DNA molecule.
  • the plasmid construct ofthe present invention contains a positive selection marker positioned between two flanking regions ofthe gene of interest.
  • the construct can also contain a screening marker, for example green fluorescent protein (GFP). If present, the screening marker is positioned outside of and some distance away from the flanking regions.
  • GFP green fluorescent protein
  • PCR refers to a method for amplifying a DNA base sequence using a heat-stable polymerase such as Taq polymerase, and two oligonucleotide primers, one complementary to the (+)-strand at one end ofthe sequence to be amplified and the other complementary to the (- )-strand at the other end. Because the newly synthesized DNA strands can subsequently serve as additional templates for the same primer sequences, successive rounds of primer annealing, strand elongation, and dissociation produce exponential and highly specific amplification ofthe desired sequence. PCR also can be used to detect the existence ofthe defined sequence in a DNA sample. "Long-range” refers to PCR conditions which allow amplification of large nucleotides stretches, for example, greater than 1 kb.
  • the term "positive selection marker” refers to a gene encoding a product that enables only the cells that carry the gene to survive and/or grow under certain conditions. For example, plant and animal cells that express the introduced neomycin resistance (Neo 1 ) gene are resistant to the compound G418. Cells that do not carry the Neo r gene marker are killed by G418. Other positive selection markers will be known to those of skill in the art.
  • Positive-negative selection refers to the process of selecting cells that carry a DNA insert integrated at a specific targeted location (positive selection) and also selecting against cells that carry a DNA insert integrated at a non-targeted chromosomal site (negative selection).
  • negative selection inserts include the gene encoding thymidine kinase (tk).
  • tk thymidine kinase
  • “Screening marker” or “reporter gene” refers to a gene that encodes a product that can readily be assayed. For example, reporter genes can be used to determine whether a particular DNA construct has been successfully introduced into a cell, organ or tissue.
  • Non-limiting examples of screening markers include genes encoding for green fluorescent protein (GFP) or genes encoding for a modified fluorescent protein. "Negative screening marker” is not to be construed as negative selection marker; a negative selection marker typically kills cells that express it.
  • the term "vector” refers to a DNA molecule that can carry inserted DNA and be perpetuated in a host cell. Nectors are also known as cloning vectors, cloning vehicles or vehicles. The term includes vectors that function primarily for insertion of a nucleic acid molecule into a cell, replication vectors that function primarily for the replication of nucleic acid, and expression vectors that function for transcription and/or translation ofthe D ⁇ A or R ⁇ A.
  • vectors that provide more than one ofthe above functions.
  • the vector contains sites useful in the methods described herein contains for example, the vectors "pDG2" or "pDG4" as described herein.
  • a "host cell” includes an individual cell or cell culture which can be or has been a recipient for vector(s) or for incorporation of nucleic acid molecules and/or proteins.
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total D ⁇ A complement) to the original parent due to natural, accidental, or deliberate mutation.
  • a host cell includes cells transfected with the constructs ofthe present invention.
  • genomic library refers to a collection of clones made from a set of randomly generated overlapping D ⁇ A fragments representing the entire genome of an organism.
  • a "cD ⁇ A library” (complementary D ⁇ A library) is a collection of all ofthe mR ⁇ A molecules present in a cell or organism, all turned into cD ⁇ A molecules with the enzyme reverse transcriptase, then inserted into vectors (other D ⁇ A molecules which can continue to replicate after addition of foreign D ⁇ A).
  • Exemplary vectors for libraries include bacteriophage (also known as "phage"), which are viruses that infect bacteria, for example lambda phage. The library can then be probed for the specific cD ⁇ A (and thus mR ⁇ A) of interest.
  • library systems which combine the high efficiency of a phage vector system with a plasmid system (for example, ZAP system from Stratagene, La Jolla, CA) are used in the practice ofthe present invention.
  • homologous recombination refers to the exchange of D ⁇ A fragments between two D ⁇ A molecules or chromatids at the site of essentially identical nucleotide sequences.
  • substantially homologous refers to polynucleotide sequences that are essentially identical. For example, homology can be determined using a "blastn” algorithm. It is understood that substantially homologous sequences can accommodate insertions, deletions, and substitutions in the nucleotide sequence. Thus, linear sequences of nucleotides can be essentially identical even if some ofthe nucleotide residues do not precisely correspond or align.
  • ligation independent cloning is used in the conventional sense to refer to incorporation of a DNA molecule into a vector or chromosome without the use of kinases or ligases. Ligation independent cloning techniques are described, for instance, in Aslanidis and de Jong, (1991) Nucleic Acids Research 18:6069-6074 and U.S. Patent Application Serial No. 07/847,298.
  • transgenic animal refers to a genetically engineered animal or offspring of genetically engineered animals.
  • the transgenic animal usually contains genetic material from at least one unrelated organism, such as from a bacteria, virus, plant, or other animal.
  • target DNA refers to the nucleic acid molecule or polynucleotide having a sequence in the general population that is not associated with any disease or discernible phenotype. It is noted that in the general population, wild-type genes may include multiple prevalent versions that contain alterations in sequence relative to each other and yet do not cause a discernible pathological effect. These variations are designated “polymorphisms” or “allelic variations.”
  • the target DNA comprises a portion of a particular gene or genetic locus in the individual's genomic DNA.
  • the target DNA comprises part of a particular gene or genetic locus in which the function of the gene product is not known, for example a gene identified using a partial cDNA sequence such as an EST.
  • exonuclease refers to an enzyme that cleaves nucleotides sequentially from the free ends of a linear nucleic acid substrate. Exonucleases can be specific for double or single stranded nucleotides and/or directionally specific, for instance, 3 '-5' and/or 5'-3'. Some exonucleases exhibit other enzymatic activities, for example, T4 DNA polymerase is both a polymerase and an active 3 '-5' exonuclease.
  • exonucleases include exonuclease III which removes nucleotides one at a time from the 5'- end of duplex DNA which does not have a phosphorylated 3 '-end, exonuclease NI which makes oligonucleotides by cleaving nucleotides off of both ends of single-stranded D ⁇ A, and exonuclease lambda which removes nucleotides from the 5' end of duplex DNA which have 5'-phosphate groups attached to them.
  • exonuclease III which removes nucleotides one at a time from the 5'- end of duplex DNA which does not have a phosphorylated 3 '-end
  • exonuclease NI which makes oligonucleotides by cleaving nucleotides off of both ends of single-stranded D ⁇ A
  • exonuclease lambda which removes nucleotides from the 5' end
  • the present invention provides novel constructs having multiple sites where 5'-3' single-stranded regions can be created. These constructs, preferably plasmids, include a vector capable of directional, four-way ligation independent cloning. By making use of these novel constructs, the present invention also offers an alternative, time-saving method for preparing a DNA construct. Examples of these constructs are shown in Figures 2 and 3.
  • the constructs typically include a sequence encoding a positive selection marker such as a gene encoding neomycin resistance; a restriction enzyme site on either side ofthe positive selection marker and a sequence flanking the restriction enzyme sites which does not contain one ofthe four base pairs.
  • a positive selection marker such as a gene encoding neomycin resistance
  • a restriction enzyme site on either side ofthe positive selection marker and a sequence flanking the restriction enzyme sites which does not contain one ofthe four base pairs.
  • a construct suitable for introducing targeted mutations into ES cells is prepared directly from a plasmid genomic library. Using long- range PCR with specific primers, a sequence of interest is identified and isolated from the plasmid library in a single step. Following isolation of this sequence, a second polynucleotide that will disrupt the target sequence can be readily inserted between two regions encoding the sequence of interest. Using this direct method a targeted construct can be created in as little as 72 hours.
  • a targeted construct is prepared after identification of a clone of interest in a phage genomic library as described in detail below.
  • any gene can be quickly and efficiently prepared for use in ES cells.
  • constructs are prepared directly from a plasmid genomic library.
  • the library can be produced by any method known in the art.
  • DNA from mouse ES cells is isolated and treated with a restriction endonuclease which cleaves the DNA into fragments.
  • the DNA fragments are then inserted into a vector, for example a bacteriophage or phagemid (e.g., Lamda ZAPTM, Stratagene, La Jolla, CA) systems.
  • a vector for example a bacteriophage or phagemid (e.g., Lamda ZAPTM, Stratagene, La Jolla, CA) systems.
  • the DNA fragments are preferably between about 5 and about 20 kilobases.
  • the organism(s) from which the libraries are made will have no discernible disease or phenotypic effects.
  • the library is a mouse library.
  • This DNA may be obtained from any cell source or body fluid.
  • Non-limiting examples of cells sources available in clinical practice include ES cells, liver, kidney, blood cells, buccal cells, cerviovaginal cells, epithelial cells from urine, fetal cells, or any cells present in tissue obtained by biopsy.
  • Body fluids include urine, blood, cerebrospinal fluid (CSF), and tissue exudates at the site of infection or inflammation. DNA extracted from the cells or body fluid using any method known in the art.
  • the DNA is extracted by adding 5 mL of lysis buffer (10 mM Tris-HCl pH 7.5), 10 mM EDTA (pH 8.0), 10 mM NaCl, 0.5% SDS and 1 mg/mL Proteinase K) to a confluent 100 mm plate of embryonic stem cells. The cells are then incubated at about 60°C for several hours or until fully lysed. Genomic DNA is purified from the lysed cells by several rounds of gentle phenolxhloroform extractions followed by an ethanol precipitation. For convenience, the genomic library can be arrayed into pools.
  • PCR Long-Range Polymerase Chain Reaction
  • a sequence of interest is identified from the plasmid library using oligonucleotide primers and long-range PCR.
  • the primers are outwardly-pointing primers which are designed based on sequence information obtained from a partial gene sequence, e.g., a cDNA or an EST sequence.
  • the product will be a linear fragment that excludes the region which is located between each primer.
  • PCR conditions found to be suitable are described below in the Examples. It will be understood that optimal PCR conditions can be readily determined by those skilled in the art. (See, e.g., PCR 2: A PRACTICAL APPROACH (1995) eds. M.J. McPherson, B.D.
  • PCR screening of libraries eliminates many ofthe problems and time-delay associated with conventional hybridization screening in which the library must be plated, filters made, radioactive probes prepared and hybridization conditions established. PCR screening requires only oligonucleotide primers to sequences (genes) of interest. PCR products can be purified by a variety of methods, including but not limited to, microfiltration, dialysis, gel electrophoresis and the like.
  • thermostable DNA polymerases are commercially available, for example, NentTM D ⁇ A Polymerase (New England Biolabs), Deep VentTM DNA Polymerase (New England Biolabs), HotTubTM DNA Polymerase (Amersham), Thermo SequenaseTM (Amersham), rBstTM DNA Polymerase (Epicenter), PfuTM DNA Polymerase (Stratagene), Amplitaq GoldTM (Perkin Elmer), and ExpandTM (Boehringer-Mannheim).
  • the direct method involves joining the long-range PCR product (i.e. the vector) and one fragment (i.e. a gene encoding a selectable marker).
  • the vector contains two different sequence regions substantially homologous to the target DNA sequence.
  • the vector also contains a sequence encoding a selectable marker, such as ampicillin.
  • the vector and fragment are designed so that, when treated to form single stranded ends, they will anneal such that the fragment is positioned between the two different regions of substantial homology to the target gene.
  • LIC Ligation independent cloning
  • LIC Ligation independent cloning
  • Single-stranded tails are created in LIC vectors, usually by treating the vector (at a digested restriction enzyme site) with T4 DNA polymerase in the presence of only one dNTP.
  • the 3' to 5' exonuclease activity of T4 DNA polymerase removes nucleotides until it encounters a residue corresponding to the single dNTP present in the reaction mix. At this point, the 5' to 3' polymerase activity of the enzyme counteracts the exonuclease activity to prevent further excision.
  • the vector is designed such that the single stranded tails created are non-complementary. For example, in the pDG2 vector, none ofthe single stranded tails ofthe four annealing sites are complementary to each other. PCR products are created by building appropriate 5 1 extensions into oligonucleotide primers.
  • the PCR product is purified to remove dNTPs (and original plasmid if it was used as template) and then treated with T4 DNA polymerase in the presence ofthe appropriate dNTP to generate the specific vector-compatible overhangs.
  • Cloning occurs by annealing ofthe compatible tails.
  • Single-stranded tails are created at the ends ofthe cloning fragments, for example using chemical or enzymatic means.
  • Complementary tails are created on the vector; however, to prevent annealing of the vector without insert, the vector tails are not complementary to each other.
  • the length ofthe tails is at least about 5 nucleotides, preferably at least about 12 nucleotides, even more preferably at least about 20 nucleotides.
  • placing the overlapping vector and fragment(s) in the same reaction is sufficient to anneal them.
  • the complementary sequences are combined, heated and allowed to slowly cool.
  • the heating step is between about 60°C and about 100°C, more preferably between about 60°C and 80°C, and even more preferably between about 60°C and 70°C.
  • the heated reactions are then allowed to cool.
  • cooling occurs rather slowly, for instance the reactions are generally at about room temperature after about an hour. The cooling must be sufficiently slow as to allow annealing.
  • the annealed fragment/vector can be used immediately, or stored frozen at - 20°C until use.
  • annealing can be performed by adjusting the salt and temperature to achieve suitable conditions.
  • Hybridization reactions can be performed in solutions ranging from about 10 mM NaCl to about 600 mM NaCl, at temperatures ranging from about 37°C to about 65°C. It will be understood that the stringency ofthe hybridization reaction is determined by both the salt concentration and the temperature. For instance, a hybridization performed in 10 mM salt at 37°C may be of similar stringency to one performed in 500 mM salt at 65°C.
  • any hybridization conditions may be used that form hybrids between substantially homologous complementary sequences.
  • a construct is made after using any of these annealing procedures where the vector portion contains the two different regions of substantial homology to the target gene (amplified from the plasmid library using long- range PCR) and the fragment is a gene encoding a selectable marker.
  • the construct After annealing, the construct is transformed into competent E. coli cells, for example DH5-alpha cells by methods known in the art, to amplify the construct. The isolated construct is then ready for introduction into ES cells.
  • a clone of interest is identified in a pooled genomic library using PCR.
  • the PCR conditions are such that a gene encoding a selectable marker can be inserted directly into the positively identified clone.
  • the marker is positioned between two different sequences having substantial homology to the target DNA.
  • Genomic phage libraries can be prepared by any method known in the art and as described in the Examples.
  • a mouse embryonic stem cell library is prepared in lambda phage by cleaving genomic DNA into fragments of approximately 20 kilobases in length. The fragments are then inserted into any suitable lambda cloning vector, for example lambda Fix II or lambda Dash II (Stratagene, La Jolla, CA)
  • pools may be created of plated libraries.
  • a genomic lambda phage library is plated at a density of approximately 1,000 clones (plaques) per plate.
  • Sufficient plates are created to represent the entire genome ofthe organism several times over. For example, approximately 1 million clones (1000 plates) will yield approximately 8 genome equivalents.
  • the plaques are then collected, for example by overlaying the plate with a buffer solution, incubating the plates and recollecting the buffer. The amount of buffer used will vary according to the plate size, generally one 100 mm diameter plate will be overlayed with approximately 4 mL of buffer and approximately 2 mL will be collected.
  • each plate lysate can be pooled at any time during this procedure and that they can be pooled in any combinations. For ease in later identification of single clones, however, it is preferable to keep each plate lysate separately and then make a pool. For example, each 2 mL lysate can be placed in a 96 well deep well plate. Pools can then be formed by taking an amount, preferably about 100 ⁇ l, from each well and combining them in the well of a new plate. Preferably, 100 ⁇ l of 12 individual plate lysates are combined in one well, forming a 1.2 mL pool representative of 12,000 clones ofthe library.
  • Each pool is then PCR amplified using a set of PCR primers known to amplify the target gene.
  • the target gene can be a known full-length gene or, more preferably, a partial cDNA sequence obtained from publicly available nucleic acid sequence databases such as GenBank or EMBL. These databases include partial cDNA sequences known as expressed sequence tags (ESTs).
  • ESTs expressed sequence tags
  • the oligonucleotide PCR primers can be isolated from any organism by any method known in the art or, preferably, synthesized by chemical means.
  • flanking regions ofthe small known region ofthe target e.g., EST
  • each flanking fragment is greater than about 1 kb in length, more preferably between about 1 and about 10 kb, and even more preferably between about 1 and about 5 kb.
  • larger fragments may increase the number of homologous recombination events in ES cells, larger fragments will also be more difficult to clone.
  • one ofthe oligonucleotide PCR primers used to amplify a flanking fragment is specific for library cloning vector, for example lambda phage.
  • primers specific for the lambda phage arms can be used in conjunction with primers specific for the positive clone to generate long flanking fragments.
  • Multiple PCR reactions can be set up to test different combinations of primers.
  • the primers used will generate flanking sequences between about 2 and about 6 kb in length.
  • the oligonucleotide primers are designed with 5' sequences complementary to the vector into which the fragments will be cloned.
  • the primers are also designed so that the flanking fragments will be in the proper 3'-5' orientation with respect to the vector and each other when the construct is assembled.
  • the cloning involves a vector and two fragments.
  • the vector contains a positive selection marker, preferably Neo r , and cloning sites on each side ofthe positive selection marker for two different regions ofthe target gene.
  • the vector also contains a sequence coding for a screening marker (reporter gene), preferably, positioned opposite the positive selection marker.
  • the screening marker will be positioned outside the flanking regions of homologous sequences.
  • Figure 3 A shows one embodiment ofthe vector with the screening marker, GFP, positioned on one side ofthe vector. However, the screening marker can be positioned anywhere between Not I and Site 4 on the side opposite the positive selection marker, Neo R .
  • a suitable vector is the plasmid vector shown in Figure 2 having the sequence of SEQ ID NO:l.
  • the specific nucleic acid ligation independent cloning sites (also referred to herein as annealing sites) labeled "sites 1 , 2, 3 or 4" in Figure 1 are also shown herein.
  • the cloning sites are lacking at least one type of base, i.e., thymine (T), guanine (G), cytosine (C) or adenine (A).
  • T thymine
  • G guanine
  • C cytosine
  • A adenine
  • T4 DNA polymerase acts as both a 3 '-5' exonuclease and a polymerase. hus, when there are insufficient nucleotides available for the polymerase activity, T4 will act as an exonuclease. Specific overhangs can therefore be created by reacting the pDG2 vector with T4 DNA polymerase in the presence of dTTP only.
  • Other enzymes useful in the practice of this invention will be known to those in the art, for instance uracil DNA glycosylase (UDG) (See, e.g., WO 93/18175).
  • UDG uracil DNA glycosylase
  • the vector exemplified herein has an overhang of 24 nucleotides. It will be known by those skilled in the art that as few as 5 nucleotides are required for successful ligation independent cloning.
  • a construct is assembled in a two-step cloning protocol.
  • each cloning region of homology is separately cloned into two ofthe anealing sites ofthe vector.
  • an "upstream” region of homology is cloned into annealing sites 1 and 2 while in a separate cloning, a "downstream" region of homology is cloned into annealing sites 3 and 4.
  • a targeting construct containing both regions of homology can be created by digesting each clone with restriction enzymes where one enzyme digests outside of annealing site 1 (e.g., Not I in figure 2A) and another enzyme digests between the positive selection marker and annealing site 3 (e.g., Sal I in figure 2A).
  • the fragments containing the flanking homology regions from each construct will be purified (e.g. by gel electrophoresis) and combined using standard ligation techniques known in the art, to produce the resulting targeting construct.
  • a construct according to one aspect ofthe present invention can be formed in a single-step, four way ligation procedure.
  • the vector and fragments are treated as described above. Briefly, the vector is treated to form two pieces, each piece having a single-stranded tail of specific sequence on each end. Likewise, the PCR amplified flanking fragments are also treated to form single-stranded tails complementary to those ofthe vector pieces. The treated vector pieces and fragments are combined and allowed to anneal as described above. Because ofthe specificity ofthe single-stranded tails, the final construct will contain the fragments separated by the positive selection marker in the proper orientation.
  • the final plasmid constructs can be used immediately for introduction into ES cells, or stored frozen ' at -20°C until use.
  • Genomic libraries using the lambda ZAPTM system were prepared as follows. Embryonic stem cells were grown in 100 mm tissue culture plates. High molecular weight genomic DNA was isolated from these ES cells by adding 5 mL of lysis buffer (10 mM tris-HCL pH 7.5, lOmM EDTA pH 8.0, 10 mM NaCl, 0.5% SDS, and 1 mg/ml Proteinase K) to a confluent 100 mm plate of embryonic stem cells. The cells were then incubated at 60°C for several hours or until fully lysed. Genomic DNA was purified from the lysed cells by several rounds of gentle phenol: chloroform extractions followed by ethanol precipitation.
  • lysis buffer 10 mM tris-HCL pH 7.5, lOmM EDTA pH 8.0, 10 mM NaCl, 0.5% SDS, and 1 mg/ml Proteinase K
  • the genomic DNA was partially digested with the restriction enzyme Sau 3A 1 to generate fragments of approximately 5-20 kb.
  • the ends of these fragments were partially filled in by addition of dATP and dGTP in the presence of Klenow DNA polymerase, creating incompatible ends on the genomic fragments. Size fragments of between 5 and 10 kb were then purified by agarose gel electrophoresis (lx TAE, 0.8%) gel).
  • the DNA was then isolated from the excised agarose pieces using a QIAquick gel extraction kit (Qiagen, Inc., Valencia, CA).
  • the genomic fragments were ligated into the Lambda ZapTM II vector (Stratagene, Inc., La Jolla, CA) that had been cut with Xho 1 and partially filled in using dTTP, dCTP, and Klenow DNA polymerase. After ligation, the DNA was packaged using a lambda packaging mix (Gigapack III gold, Stratagene, Inc., La Jolla, CA) and the titer was determined.
  • Lambda ZapTM II vector Stratagene, Inc., La Jolla, CA
  • a lambda packaging mix Gigapack III gold, Stratagene, Inc., La Jolla, CA
  • Circular phagemid DNA was derived from the lambda library by growing the lambda clones on the appropriate bacterial strain (XL-1 Blue MRF, Stratagene, Inc.) in the presence ofthe Ml 3 helper phage, ExAssist (Stratagene, Inc.). Specifically, approximately 100,000 lambda clones were incubated with a 10-100 fold excess of both bacteria and helper phage for 20 minutes at 37 °C. One ml of LB media + 1 OmM MgSO Register was added to each excision reaction and it was incubated overnight at 37°C with shaking. Typically 24- 96 of these reactions were set up at a time in a 96 well deep-well block.
  • the block was heated to 65 °C for 15 minutes to kill both the bacteria and the lambda phage. Bacterial debris was removed by centrifugation at approximately 3000g for 15 minutes. The supernatant containing the circular phagemid DNA, was retained and used directly in plasmid PCR experiments (see Examples 9 and 10 for plasmid PCR experiments).
  • the pools of phagemid DNA described above were screened for specific genes of interest using long-range PCR and "outward pointing" oligos, chosen as described above based on the known sequence (depicted in Figure 1).
  • the PCR reactions contains 2 ⁇ l of a pool phagemid DNA sample, 3 ⁇ l of l Ox PCR Buffer 3 (Boehringer Mannheim), 1.1 ⁇ l 10 mM dNTPs, 50 nM primers, 0.3 ⁇ l of EXPAND Long Template PCR Enzyme Mix (Boehringer-Mannheim) and 30 ⁇ l of H 2 0. Cycling conditions were 94°C for 2 minutes (1 cycle); 94°C for 10 seconds, 65°C for 30 seconds, 68°C for 15 seconds (15 cycles); 94°C for 10 seconds, 60°C for 30 seconds, 68°C for 15 seconds plus 20 seconds increase per each additional cycle (25 cycles); 68°C for 7 minutes (1 cycle) and holding at 4°C.
  • the products ofthe PCR reactions were separated by electrophoresis through agarose gels containing IX TAE buffer and visualized with ethidium bromide and UN light. Any large fragments indicative of successful long-range PCR were excised from the gel and purified using QlAquick PCR purification kit (Qiagen).
  • PCR fragments were generated by mixing: 0.1-2 ⁇ g ofthe fragment; 2 ⁇ l of ⁇ EB (New England BioLabs) Buffer 4; 1 ⁇ l of 2 mM dTTP, 6 units of T4 polymerase (NEB), H 2 O to total volume of 20 ⁇ l and incubating at 25°C for 30 minutes. The polymerase is inactivated by heating at 75°C for 20 minutes.
  • Single- stranded ends were also created on the Neo R selectable marker fragment by digesting the plasmid vector pDG2 at the unique restriction sites, with Sad and SacII (pDG2 depicted in Figure 2A) and treating each reaction with T4 polymerase as above.
  • the vector shown in Figure 1 was prepared with single-stranded ends complementary to those on the long range PCR fragment. The vector and fragments were then assembled into constructs using either a two- step cloning strategy or a four- way, single-step protocol.
  • a reaction containing 10 ng of T4 treated NEO cassette, 2 ⁇ l of T4-treated PCR fragment, 0.2 ul of 0.5 M EDTA, 0.3 ⁇ l of 0.5 M NaCl and H 2 O up to 4 ⁇ l was heated to 65°C and allowed to cool to room temperature over approximately 45 minutes. The mixture was then transformed into subcloning DH5- ⁇ efficiency competent cells.
  • a mouse embryonic stem cell library was prepared in lambda phage as follows. Genomic libraries were constructed from genomic DNA by partial cleavage of DNA at Sau 3A1 sites to yield genomic fragments of approximately 20 kb in length. The terminal sequences of these DNA fragments were partially filled in using Klenow enzyme in the presence of dGTP and dATP and the fragments were ligated using T4 DNA ligase into Xho I sites of an appropriate lambda cloning vector, e.g., lambda Fix II (Stratagene, Inc., La Jolla, California), which had been partially filled in using Klenow in the presence of dTTP and dCTP.
  • an appropriate lambda cloning vector e.g., lambda Fix II (Stratagene, Inc., La Jolla, California), which had been partially filled in using Klenow in the presence of dTTP and dCTP.
  • the partially digested genomic DNA was size selected using a sucrose gradient and sequences of approximately 20 kb selected for.
  • the enriched fraction was cloned into a Bam HI cut lambda vector, e.g., lambda Dash II (Stratagene, Inc., La Jolla, California).
  • the library was plated onto 1,152 plates, each plate containing approximately 1 ,000 clones. Thus, a total of 1.1 million clones (the equivalent of 8 genomes) was plated.
  • the phage were eluted from each plate by adding 4 mL of lambda elution buffer (10 mM MgCl 2 , 10 mM Tris-pH 8.0) to each plate and incubating for 3 to 5 hours at room temperature. After incubation, 2 mL of buffer was collected from each plate and placed into one well of a 96 deep well plate (Costar, Inc.). Twelve 96-well plates were filled and referred to as the "sub-pool library.”
  • pool libraries were made by placing 100 ⁇ l of 12 different sub-pool wells into one well of a new 96 well plate. The 12 sub-pool plates were combined to form 1 plate of pool libraries.
  • PCR was performed in the presence of 0.5 units of Amplitaq GoldTM (Perkin Elmer), 1 ⁇ M of each oligonucleotide, 200 ⁇ M dNTPs, 2 ⁇ l of a 1 to 5 dilution ofthe pool (or subpool) supernatant, 50 mM KC1, 100 mM Tris-HCl (pH 8.3), and either 1.5 mM or 1.25 mM MgCl 2 .
  • Amplitaq GoldTM Perkin Elmer
  • 1 ⁇ M of each oligonucleotide 200 ⁇ M dNTPs
  • 2 ⁇ l of a 1 to 5 dilution ofthe pool (or subpool) supernatant 50 mM KC1, 100 mM Tris-HCl (pH 8.3), and either 1.5 mM or 1.25 mM MgCl 2 .
  • knock-out constructs contain two blocks of DNA sequence homologous to the target gene, flanking a positive selection marker.
  • Long range PCR was performed from the pools of lambda clones positively identified as described above in Example 2. Each fragment was generated using a pair of oligonucleotides with predetermined sequences lacking one type of base and complementary to predetermined sequences on the vector. The fragments obtained were between 1 and 5 kb. A third fragment, longer than 5 kb, is also generated using appropriate oligonucleotides. This third fragment was then used to obtain DNA sequences near the gene to be knocked out but outside ofthe vector.
  • the pDG2 plasmid vector ( Figure 2A) contains unique restriction sites SacII and Sac I. Appropriate single-stranded annealing sites were generated by digesting the pDG2 vector with either restriction enzyme SacII or Sad and treating each reaction with T4 polymerase and dTTP as described above.
  • reaction containing 1 ⁇ l of T4-treated vector, 0.2 ⁇ l of 0.5M EDTA, 3 ⁇ l of 0.5M NaCl and 0.5 ⁇ l H 2 O and 1 ⁇ l of either (1) T4 polymerase-treated fragments; (2) a 1 : 10 dilution of the T4-treated fragments reaction; (3) a 1 : 100 dilution of the T4- treated fragments or (4) H 2 O (no insert control).
  • the microtiter plates were sealed, placed in-between two temperature blocks heated to 65°C, and allowed to cool slowly at room temperature for 30 to 45 minutes.
  • microtiter plate was then placed on ice and 20-25 ⁇ l of subcloning efficiency competent cells added to each well. The plate was incubated on ice for 20-30 minutes.
  • microtiter plate was then placed between two temperature blocks heated to 42°C for 2 minutes, followed by 2 minutes on ice.
  • 100 ⁇ l of LB was added to each well, the plate covered with parafilm and incubated 30-60 minutes at 37°C.
  • the entire contents of each well were plated on one LB-Amp plate and incubated at 37°C overnight. Between about 12-24 colonies were picked from plates which had at least 2-4 times more colonies than the no insert control. The colonies were grown in deep well plates overnight at 37°C and then the plasmid DNA extracted using a Qiagen mini-prep kit.
  • the plasmid DNA was digested with Not I and Sal I enzymes. As shown in Figure 2A, a Not I/Sal I digestion will generate a large fragment containing cloning sites 3 and 4 and a smaller fragment containing cloning sites 1 and 2 and the Neo r gene. After digestion, the reactions were run on a 0.8% agarose gel containing 0.2 ⁇ g/mL ethidium bromide. For no inserts, two bands were present, one of 1975 base pairs and one of 2793 base pairs. When an insert fragment was present, at least one of these bands would be larger because it would also contain a fragment (insert 1 or 2) either at the annealing site 1/2 or the site 3/4.
  • the insert bands were excised and treated with a QlAquick gel extraction kit.
  • a second ligation reaction was performed containing 1 ⁇ l of 10X ligase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 10 mM dithiothreitol, 1 mM ATP, 25 ⁇ g/mL bovine serum albumin), 1 ⁇ l T4 DNA ligase, 1-2 ⁇ l fragment (site 3/4 band), 5 ⁇ l of site 1/2 band and H 2 O up to 10 ⁇ l. Controls were also set up replacing either the site 3/4 fragment or the site 1/2 fragment with water. The reactions were incubated 1 to 2 hours at room temperature and transformed with 25 ⁇ l of competent cells.
  • RI genomic library refers to a genomic library prepared from the Rl ES cell line. Such libraries can be prepared such as described in Example 1. To date, the methods ofthe invention have been practiced in about 200 known and novel target genes.
  • Example 4 Two-way Cloning of Targeting Construct for Target 2, a Metalloprotease Gene
  • Pool C2 was then amplified using oligos 454 (SEQ ID NO:21) and 463 (SEQ ID NO :22) to generate a 2000 bp band
  • pool H2 was amplified using oligos 464 (SEQ ID NO:23) and 42 (SEQ ID NO:24) to generate a 2700 bp band. These two bands contained flanking DNA for target 2.
  • Each band containing flanking DNA for target 2 was gel purified from an agarose gel and the ends were treated individually with T4 DNA polymerase in the presence of dTTP in order to produce single stranded overhangs. Each of these bands was then cloned individually into plasmid vector pDG2 (shown in Figure 2A).
  • the C2 band was cloned into Sac Il-digested pDG2 that had been treated with T4 DNA polymerase in the presence of dATP, by ligation independent cloning.
  • the H2 band was cloned into Sac I-digested pDG2 that had been treated with T4 DNA polymerase in the presence of dATP by ligation independent cloning.
  • each vector above was digested with Not I / Sal I and the 4 kb fragment containing the C2 band and the 5 kb fragment containing the H2 band were gel purified. These two fragments were ligated together with T4 DNA ligase using standard conditions, and recombinants containing both flanking arms were identified. Out of 12 colonies examined, all 12 were correct, i.e. contained both arms correctly flanking the positive selection marker, Neo R .
  • Example 5 Two-way Cloning of Targeting Construct for Target 54, a Serine Protease Gene
  • flanking DNA for target 54 Individual pools of an Rl genomic library were PCR amplified under standard conditions using oligos #151 (SEQ ID NO:25) and #155 (SEQ ID NO:26) in order to identify individual wells containing genomic DNA of target #54 as indicated by the presence of a 179 bp band. A total of 12 pools, each containing approximately 12,000 clones were identified (pools A4, A10, B2, B9, C9, El, E6, F8, G4, H6, H7, and H9).
  • Pool G4 was then amplified using oligos 454 (SEQ ID NO:27) and 465 (SEQ ID NO:28) to generate a 1400 bp band and pool H7 was amplified using oligos 466 (SEQ ID NO:29) and 42 (SEQ ID NO:24) to generate a 3000 bp band. These two bands contained flanking DNA for target 54.
  • Each band was gel purified from an agarose gel and the ends were treated individually with T4 DNA polymerase in the presence of dTTP in order to produce single stranded overhangs. Each of these bands were then cloned individually into pDG2.
  • the G4 band was cloned into Sac II cut pDG2 that had been treated with T4 DNA polymerase in the presence of dATP by ligation independent cloning.
  • the H7 band was cloned into Sac I cut pDG2 that had been treated with T4 DNA polymerase in the presence of dATP by ligation independent cloning.
  • each vector above was digested with Not I / Sal I and the 6 kb fragment containing the G4 band and the 8 kb fragment containing the H7 band were gel purified. These two fragments were ligated together with T4 DNA ligase using standard conditions and recombinants containing both flanking arms were identified. Out of 24 colonies examined, 14 had the correct inserts.
  • Example 6 Single-step (Four- Way) Cloning - General Procedure
  • each single-stranded annealing site is unique, a four- way ligation strategy was also used to generate constructs in a single step.
  • the annealing reactions were set up as described above except that each reaction contained a vector digested with both Sacl and SacII, and both T4-treated fragments were added to these reactions.
  • Example 7 Four- ay Cloning of Targeting Construct for Target 43, a Gene for a G- protein Coupled Receptor
  • Pool El was then amplified using oligos 41 (SEQ ID NO:32) and 38 (SEQ ID NO:33) to generate a 1500 bp band and pool D10 was amplified using oligos 40 (SEQ ID NO:34) and 37 (SEQ ID NO:35) to generate a 3500 bp band. These two bands contained flanking DNA for target 43.
  • Each band was gel purified from an agarose gel and the ends were treated individually with T4 DNA polymerase in the presence of dTTP in order to produce single stranded overhangs.
  • These inserts were then mixed with ⁇ 50ng of pDG2 that had been digested with both Sac I and Sac II followed by treatment with T4 DNA polymerase in the presence of dATP.
  • the DNA mixture was heated to 65 °C for 2 minutes followed by a 5 minute incubation on ice.
  • the annealed DNA was then transformed into competent DH5 ⁇ cells and recombinant molecules were obtained by selection on ampicilin agarose plates. After incubation overnight at 37°C, individual colonies were picked and grown up for analysis. Recombinant molecules were identified by appropriate restriction enzyme digestion. Out of 52 colonies examined, 35 had the correct restriction pattern for the expected product.
  • Example 8 Four-way Cloning of Targeting Construct for Target 244, a Novel Gene
  • Pool G9 was then amplified using oligos 445 (SEQ ID NO:38) and 667 (SEQ ID NO:39) to generate a 1300 bp band and pool A6 was amplified using oligos 668 (SEQ ID NO:40) and 42 (SEQ ID NO:24) to generate a 1600 bp band. These two bands contained flanking DNA for target 244.
  • the isolated DNA fragment was treated with T4 DNA polymerase in the presence of dTTP in order to generate appropriate single-stranded ends.
  • This fragment was then annealed (ligation independent) with a neo gene fragment obtained from pDG2 that had been digested with both Sac I and Sac II followed by treatment with T4 DNA polymerase in the presence of dATP.
  • the digestion and polymerase treatment yielded a neo gene with ends that would specifically anneal to the target 227 fragment. Annealing reactions were set up essentially as described above and a target 227 construct was obtained (13 out of 14 clones were correct).
  • Example 10 Plasmid PCR Method of Cloning Targeting Construct for Target 125, a Nuclear Hormone Receptor Gene
  • the isolated DNA fragment was treated with T4 DNA polymerase in the presence of dTTP in order to generate appropriate single-stranded ends.
  • This fragment was then annealed with a neo gene fragment obtained from pDG2 that had been digested with both Sac I and Sac II followed by treatment with T4 DNA polymerase in the presence of dATP. This yielded a neo gene with ends that would specifically anneal to the target 125 fragment. Annealing reactions were set up essentially as described above and a target 125 construct was obtained (12 out of 18 clones were correct).
  • GFP Green Fluorescent Protein
  • the respective targeting vector was linearized with a restriction endonuclease and 20 ⁇ g of DNA was added to 10 x 10 6 ES cells in ES medium ⁇ High Glucose DMEM (without L-Glutamine or Sodium Pyruvate) with LIF (Leukemia Inhibitory Factor-Gibco 13275-029 "ESGRO”) 1,000 units/ml, and 12% Fetal Calf Serum ⁇ .
  • Cells were placed into a 2 mm gap cuvette and electroplated on a BTX electroporator at 400 ⁇ F resistance and 200 volts. Immediately after electroporation, ES cells were plated at 1 x 10 6 cells per 100mm gelatinized tissue culture plate.
  • Table 1 shows the data for 3 targeted genes where ES colonies were previously tested for homologous recombination without a GFP gene marker; in two cases no homologous integrants were found. For the third gene, only 1 recombinant was found in 907 ES colonies that were tested. The GFP gene was then inserted in the targeting vector outside the region of homology and the experiments were repeated as described above. After selecting only ES colonies that do not express the GFP, homologous recombinants were found for all 3 genes. The enrichment was 4-5 fold, thus decreasing substantially the number of colonies that must be screened. In Table 2, data is presented in which a fourth gene was targeted with a knock-out construct containing a GFP screening marker.
  • the colonies that are non-flourescent After the colonies that are non-flourescent have been identified, they are picked into 96-well plates with trypsin. After the colonies have been in the typsin for 5-10 minutes the cells are divided into duplicate plates containing ES medium (one plate to freeze and one plate from which to make DNA to screen the colonies for homologous recombination events). The plate for freezing is typically grown for 2-5 days before it is frozen (freeze media: 50% FBS, 40% DMEM and 10% DMSO).
  • the DNA plate is typically overgrown and refed for 8-10 days before it is lysed to prepare DNA for PCR or Southern blot analysis (lysis buffer: lOmM TRIS pH 7.5, lOmM EDTA pH 8.0, lOmM NaCl, 0.5% sarcosyl and lmg/ml Proteinase K).
  • lysis buffer lOmM TRIS pH 7.5, lOmM EDTA pH 8.0, lOmM NaCl, 0.5% sarcosyl and lmg/ml Proteinase K.
  • the positive well(s) is thawed into a 24-well tissue culture dish that has been previously plated with mitomycin C treated mouse embryonic fibroblasts (24 hours prior).
  • the cells are grown up to sufficient levels for diploid aggregation (CD-I host strain) or blastocyst injection (C57BL/6 host strain) and also for additional freezing of stock vials.
  • CD-I host strain diploid aggregation
  • blastocyst injection C57BL/6 host strain
  • additional freezing of stock vials For general procedures for the handling of ES cells and the production of chimeric mice from ES cells, refer to Teratocarcinomas and Embryonic Stem Cells-a Practical Approach (Ed. EJ Robertson, IRL Press Limited, 1987).
  • the blastocysts are then implanted in pseudo pregnant female CD-I mice. Offspring are born 17-20 days later. Highly chimeric mice are then bred to produce germline transmission ofthe mutated gene.

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Abstract

L'invention concerne de nouvelles constructions nucléotidiques, ainsi que des méthodes permettant de produire des constructions d'ADN utilisées pour introduire des séquences dans un gène de cellule, en particulier de cellule souche embryonnaire, ou pour interrompre ledit gène.
PCT/US2000/018812 2000-07-11 2000-07-11 Methodes permettant de creer des constructions utilisees pour introduire des sequences dans des cellules souches embryonnaires WO2002004621A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/US2000/018812 WO2002004621A2 (fr) 2000-07-11 2000-07-11 Methodes permettant de creer des constructions utilisees pour introduire des sequences dans des cellules souches embryonnaires
AU2000260840A AU2000260840A1 (en) 2000-07-11 2000-07-11 Methods of creating constructs useful for introducing sequences into embryonic stem cells

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2000/018812 WO2002004621A2 (fr) 2000-07-11 2000-07-11 Methodes permettant de creer des constructions utilisees pour introduire des sequences dans des cellules souches embryonnaires

Publications (2)

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WO2002004621A2 true WO2002004621A2 (fr) 2002-01-17
WO2002004621A3 WO2002004621A3 (en) 2002-04-25

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PCT/US2000/018812 WO2002004621A2 (fr) 2000-07-11 2000-07-11 Methodes permettant de creer des constructions utilisees pour introduire des sequences dans des cellules souches embryonnaires

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AU (1) AU2000260840A1 (fr)
WO (1) WO2002004621A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1373473A1 (fr) * 2001-12-04 2004-01-02 Genome Biosciences, LLC Procedes et vecteurs de ciblage genetique

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0663952A4 (fr) * 1992-09-11 1997-06-11 Univ California Animaux transgeniques possedant des genes de transduction lymphocytaire cibles.
US5714667A (en) * 1995-11-06 1998-02-03 Amgen Canada Inc. Mice lacking expression of CTLA-4 receptor
IL149277A0 (en) * 1999-10-26 2002-11-10 Deltagen Inc Transgenic mice containing trp gene disruptions
EP1304920A2 (fr) * 2000-03-16 2003-05-02 Deltagen, Inc. Souris transgeniques contenant des interruptions de genes ciblees

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1373473A1 (fr) * 2001-12-04 2004-01-02 Genome Biosciences, LLC Procedes et vecteurs de ciblage genetique
EP1373473A4 (fr) * 2001-12-04 2005-02-16 Genome Biosciences Llc Procedes et vecteurs de ciblage genetique

Also Published As

Publication number Publication date
AU2000260840A1 (en) 2002-01-21
WO2002004621A3 (en) 2002-04-25

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