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  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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  • 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
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  • A01K2217/00—Genetically modified animals
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  • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
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• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/03—Animal model, e.g. for test or diseases

Definitions

• the field of this invention concerns the genomic modification of vertebrate genes by defined deletions of DNA.
• transgenic animal technology can be utilized to permanently change the germline DNA of an entire animal.
• the genome can be manipulated to either add or delete DNA sequences.
• a series of deletions may be made, where a variable amount of DNA is removed by holding one end-point constant, and varying the position of the second end- point. Such a series may be used for the experimental mapping of genes, as there is interest in determining a genetic map for animals, particularly humans.
• Some DNA sequences act as negative regulators for gene expression. It may be possible to activate the expression of certain genes by deleting those upstream or downstream elements which are responsible for silencing a gene using defined deletions of DNA to remove weak promoters or enhancers, and insert stronger foreign promoters or enhancers near the transcriptional initiation site of a native gene. Alternatively, stronger promoters or enhancers may be moved from a wild type gene located upstream near the target gene.
• the replacement targeting vector comprises markers for positive and negative selection, homologous regions for targeting at a specific site, and an additional homologous region to mediate the deletion.
• Figure 1 is a diagram of a targeting construct and target chromosome, as described in the Example, infra.
• Figure 2 is a diagram of a bidirectional targeting construct and target chromosome, as described in the Example, infra.
• Figure 3 is a diagram of successive stages of deletion, as described infra.
• Figure 4 is a diagram of the targeting construct for inactivation of the immunoglobulin kappa light chain J and Constant regions and design of the targeting experiment described in the Example, infra.
• Figure 5 is a diagram of the construction of vectors for inactivating the immunoglobulin kappa light chain J and constant regions as described in the Example, infra.
• Figure 6 is a diagram of the final deletion vectors for inactivation of the immunoglobulin kappa light chain J and Constant regions as described in the Example, infra.
• Figure 7 is an illustration of the Southern analysis of light chain J and Constant region deleted cells as described in the Example, infra.
• a DNA construct for homologous targeting via replacement of DNA are used to introduce a defined lesion at a specific site in a vertebrate chromosome.
• lesion is meant an alteration in the DNA sequences at the site.
• the targeting construct may be used to delete large segments of chromosomal DNA, without leaving vector DNA sequences in the targeted chromosome.
• the method may also be used to alter a sequence in the target chromosome, without leaving behind vector DNA sequences.
• the targeting construct consists of a positive selectable marker, a negative selectable marker, which in some instances may be the same marker, sequences with homology to the target site in the chromosome, and an additional homologous sequence from a region outside of the sequences to be targeted in the chromosome. Therefore, the target site in the chromosome is defined by the homologous sequences in the vector.
• the additional homologous DNA sequence (ADH additional DNA homology) is homologous to a sequence on the same chromosome as the target sequences, which additional sequence can be located either upstream or downstream on the chromosome. The sequence of DNA which lies between the ADH sequence and the target sequences on the chromosome will ultimately be deleted.
• a preferred targeting construct for use in the invention is an "omega" or "replacement” targeting vector.
• an "O" or “insertional” targeting vector may also be used in some situations where a single target sequence is sufficient for providing homologous sequences for recombination.
• the targeting construct is introduced into a host cell, preferably after linearization, where it undergoes homologous recombination to integrate at the site of the target sequences.
• the arrangement of elements in the linear construct is shown in Figure 1.
• an exemplary order of functional units is shown, it should be understood that the order of the functional sequences between the target sequences may be arranged in other ways, depending on the particular use contemplated.
• the chromosomal sequences that are to be targeted by the homologous sequences in the targeting construct may be contiguous or separated. Where separated, homologous recombination in a double cross-over event will result in an initial deletion.
• v-ADH refers to homologous sequences in the chromosome or vector used for effecting the defined deletion.
• a double crossover event at the target sites in the chromosome results in the replacement of the chromosomal region lying between the two c-target sequences, unless the c-target sequences are contiguous, with the construct sequences lying between the two v-target arms of homology.
• Sequences which are not functional in the vertebrate host, such as bacterial origins of replication and antibiotic resistance may be outside or inside of the construct arms. Sequences located outside of the arms will not be integrated into the chromosome, while those sequences between the arms will be integrated.
• the v-ADH region and selectable markers for vertebrate cells will be positioned between the two arms of homology.
• the positive selectable marker allows one to apply selective pressure, and to grow only those cells which contain the integrated construct.
• An illustration of the resulting chromosome with integrated construct is shown in Figure 1 as the "Targeted chromosome".
• the illustration of the "Chromosome” shows the c-target sequences as contiguous, although it should be understood that they may be separated on the chromosome, as described earlier.
• One copy of each target sequence and two copies of the ADH sequence will remain.
• One ADH copy is chromosomal in origin (c-ADH)
• the other ADH sequence is derived from the targeting construct (v- ADH) .
• the two copies are separated by the chromosomal sequences and at least the negative selectable marker of the targeting construct that lie between the v-ADH and c- ADH sequences.
• the cells Once the cells have been positively selected they can be directly negatively selected by the addition of negative selection media to the cells, for example using gancyclovir when the marker is the Herpes thymidine kinase gene. Under negative selection conditions, cells that retain the negative selectable marker positioned between the target sequences, will usually die. Cells that have lost the negative selectable marker because of an intramolecular crossover event involving the homologous c- ADH and v-ADH sequences are able to proliferate under negative selection conditions.
• the positively selected cells may be screened to identify clones targeted by homologous recombination and the targeted clones can be subjected to negative selection.
• a second independent stage of negative selection may be needed depending on the absolute efficiencies of the initial intermolecular homologous recombination step and of the deletion resulting from the intramolecular crossover.
• the method of the invention may also employ negative selection using a different negative selectable marker from that positioned between the v- target sequences to increase the efficiency of the intermolecular homologous targeting step.
• the deletion is an intramolecular recombination event, and will have a higher efficiency than homologous recombination which is intermolecular.
• the deletion event contemplated in this invention thus has a much higher probability of providing large deletions of DNA than using intermolecular homologous recombination directly.
• FIG. 1 An example of the order of elements in the construct is depicted in Figure 1.
• the 5' and 3' ends will usually be bounded by the v-target sequences.
• the v-ADH sequence and the selectable markers will lie between the v-target sequences.
• the v-target and v-ADH sequences will all be in the same orientation, that is, the direction of the DNA of the individual components will be the same in the construct as it is on the chromosome, either 5' to 3', or 3' to 5' .
• the negative selectable marker transcriptionally oriented in either direction, will lie between the v-ADH sequence, and one of the v-target sequences in the construct. The exact position will depend on the position of the c-ADH sequence. On the targeted chromosome, the negative marker lies between the c-ADH and the v-ADH.
• the position of the positive selectable marker on the construct will depend on the desired final product, because the excision step may or may not leave behind the positive marker in the chromosomal locus. If the positive and negative selectable markers are both positioned between the c-ADH and the v-ADH in the targeted chromosome, then both will be deleted upon application of negative drug selection. If the negative selectable marker is positioned between the c-ADH and v-ADH - and the positive marker is outside, then the excision step will leave the positive marker behind in the chromosome of the deleted locus. In this situation, it may be preferred to apply both positive and negative selection during the excision step. In general, the positive selectable marker may also be oriented in either transcription direction. As with the positive selectable marker, any exogenous sequences from the construct which do not lie between the c-ADH and the vADH in the targeted chromosome will remain at the locus after the final recombination event.
• the arrangement of elements on the chromosome will be as shown in Figure 1.
• the two c-target sequences will be adjacent to each other to facilitate the initial double crossover event or may be separated to provide for a deletion upon homologous recombination.
• the distance between the c-ADH and c- target sequences on the chromosome will depend on the desired final product. Normally this distance will be much larger than the distance between the two c-target sequences.
• the length of homologous v-target and v-AnH sequences will be at least about 50 nucleotides in length, preferably at least about 100 nucleotides, usually at least about 0.5 kb in length, and less than about 100 kb in length, usually less than about 20 kb, and preferably less than about 1 kb.
• the regions of homology will have at least about 90%, preferably at least about 95%, and more preferably at least about 99%, sequence identity with the sequence of the native chromosome.
• the v-target sequences may provide for subtle changes in the region of homology, such as introduction of a restriction site, changing the sequence of an enhancer or promoter locus, introducing or removing a splice site, or the like.
• subtle change is intended fewer than a total of 10 bp substitutions and/or deletions, where the percentage of base pairs involved in the target sequence does not exceed 10%, usually does not exceed 5%.
• markers may be used for selection. These include the HPRT minigene (Reid, et al. (1990) Proc. Natl. Acad. Sci. 87:4299-4303), the neo gene for resistance to G418, the HSV thymidine kinase gene for sensitivity to gancyclovir, the hygromycin resistance gene, etc.
• one may use separate markers for positive selection, such as neo. hygromycin resistance, etc. and for negative selection, such as HSV-TK, cytosine deaminase, etc.
• Other elements of the construct may include sequences which code for specific primer regions which may be used in the polymerase chain reaction (PCR) to identify recombinants, the addition of one or more restriction sites which allow for identification by gel electrophoresis, the removal of a restriction site at the target locus, or other modification which allows for identification of target cells which have undergone the desired modification.
• Various techniques may be used to introduce the linear DNA into the target cell. Techniques include electroporation, calcium precipitated DNA, fusion, transfection, lipofection, and the like. The particular manner in which the DNA is introduced into the cell is not critical to this invention, although electroporation is preferred.
• the cells may then be selected by means of the marker gene by plating in a selective medium, growth in selective culture, or Southern analysis.
• the cells may also be analyzed using PCR by employing primers which will provide for different sized fragments depending upon whether homologous recombination has occurred. In this way, target cells which have undergone the desired modification may be identified.
• Modified cells may also be identified by changes in gene expression, if appropriate. If a deletion will remove a negative regulatory element, or silencer, then one would expect increased gene transcription and protein synthesis. Alternatively, if a gene or positive regulatory element, such as an enhancer, is deleted, then one would expect to find loss of transcription and protein synthesis.
• the presence or absence of the gene product may be detected by using specific antibodies, by functional assays for the gene product, by detecting the absence or presence of mRNA from the gene, or the like. If the gene product is a surface membrane protein, one may use monoclonal antibodies in conjunction with FACS for identification of cells with the desired phenotype.
• the subject methodology may by used for mammalian fine-structure genetic analysis, investigation of lesions implicated in genetic diseases and manipulation of gene expression.
• Genes which may be targeted include beta- globin, enzymes of erythrocyte metabolism, the complement system, coagulation factors, dystrophin, enzymes of carbohydrate, lipid, amino acid, steroid, purine and pyrimidine metabolism, transport proteins, such as cystic fibrosis transmembrane regulator, immunoglobulin genes, T cell receptor genes, histocompatibility antigens, both major and minor, and the like.
• genes may be modified by inactivating loci, where one wishes to inactivate a particular combination of exons, as are present in the immunoglobulin loci, such as the heavy and/or light chain (kappa and lambda) loci, for example to eliminate the ability of a host animal to produce endogenous immunoglobulin heavy and/or light chains.
• immunoglobulin loci such as the heavy and/or light chain (kappa and lambda) loci, for example to eliminate the ability of a host animal to produce endogenous immunoglobulin heavy and/or light chains.
• MHC antigens include surface major histocompatibility complex (MHC) antigens, for transplantation.
• MHC surface major histocompatibility complex
• a portion or substantially all of the MHC can be deleted using the method of the invention.
• the Class I and Class II MHC antigens are heterodimers, each consisting of an and a jS subunit.
• the ⁇ subunit is ⁇ 2 - microglobulin.
• deletion of other genes that affect MHC antigen expression may be accomplished using the method.
• genes that regulate MHC antigen expression such as the TAP1, TAP2 genes, LMP2 and LMP7 in the Class II locus that regulate MHC antigen dependent presentation, may be deleted to prevent MHC presentation on mammalian cells.
• the cell lacking at least one competent MHC antigen may find use as a donor to an allogeneic host or if an embryonic stem cell, may find use in the production of chimeric mammalian hosts which themselves could be used as a source of organs for transplantation.
• the method involves transfection of mammalian cells, particularly normal cells, of a predetermined species with the targeting construct of the invention for deleting DNA in one of the loci related to the /3 2 -microglobulin gene, the ⁇ -subunit( ⁇ ) of the Class I or II MHC antigens, the ⁇ - subunit(s) of the Class II MHC antigens, or the genes associated with regulation of expression of the MHC antigens.
• the targeting construct will create a deletion resulting in deletion in at least one, usually both copies, of the native gene(s) , so as to prevent expression of a functional MHC antigen molecule.
• the cells having a single unmutated copy of the target gene are amplified and may be subjected to a second transformation, where the deletion may be the same or different from the first lesion.
• the resulting transformants are screened for the absence of a functional target antigen and the DNA of the cell may be further screened to ensure the absence of a wild-type target gene.
• homozygosity as to a phenotype may be achieved by breeding hosts heterozygous for the mutation.
• the cells which may be subjected to transformation may be any mammalian cells of interest, which may find use in cell therapy, research, interaction with other cells in vitro or the like.
• Cells of particular interest include, among other lineages, the islets of Langerhans, adrenal medulla cells which may secrete dopamine, osteoblasts, osteoclasts, epithelial cells, endothelial cells, T- lymphocytes, neurons, glial cells, ganglion cells, retinal cells, embryonic stem cells, liver cells, bone marrow cells, and myoblast (muscle) cells.
• the cells may be obtained from any mammalian host, including murine and other rodents, lagomorphs, porcine, feline, bovine, canine, human, etc.
• Cells from bare lymphocyte syndrome patients may be isolated in accordance with conventional ways, e.g. , panning, affinity columns, magnetic beads, or the like.
• monoclonal antibodies specific for the lymphoid cell type, B- or T-cell using monoclonal antibodies for such markers as CD 3, 4, 8, 10, 15 or 19, the desired group of cells and their progenitors may be isolated in a substantially homogeneous composition.
• the genetically defective cells may be used in the same manner as MHC antigen defective cells produced by homologous recombination.
• the MHC antigen deficient cells will be selected to achieve a particular function and be introduced into a mammalian host or used for research or other purpose.
• the stem cells which act as the progenitors for any of the above cells, which may be the original progenitor or a progenitor cell which is already dedicated to a particular lineage.
• epidermal cells such as keratinocytes, retinal epithelial cells, myoblasts, he atopoietic cells, and other cells which may be readily manipulated in vitro, maintained for long periods of time in culture and may be introduced into a host, where the cells will remain viable and functional for long periods of time.
• an embryonic stem cell line may be employed or embryonic stem cells may be obtained freshly from a host such as a murine animal, e.g. a mouse, rat, guinea pig, Chinese hamster or other small laboratory animals.
• the cells may be grown on an appropriate fibroblast-feeder layer or grown in the presence of leukemia inhibiting factor (LIF) and then used for mutation.
• LIF leukemia inhibiting factor
• the targeting constructs of the invention may be modified to include functional entities that may find use in the preparation of the construct, amplification, transformation of the host cell, and integration of the construct into the host cell.
• Techniques which may be used include calcium phosphate/DNA coprecipitates, microinjection of DNA into the nucleus, electroporation, bacterial protoplast fusion with intact cells, transfection, or the like.
• the DNA may be single or double stranded, linear or circular, relaxed or supercoiled DNA.
• Methods in Enzymolocrv (1990) 185:527-537 See Keown et aJ . , Methods in Enzymolocrv (1990) 185:527-537.
• any convenient technique for introducing the DNA into the target cells may be employed. After transformation of the target cells, many target cells are selected by means of positive and/or negative markers, as previously indicated, neomycin resistance and Acyclovir or Gancyclovir resistance. Those cells which show the desired phenotype may then be further analyzed by restriction analysis, electrophoresis,
• the resulting transformed cells may then be selected by the absence of the target MHC antigen on the surface of the cell.
• This can be achieved in a variety of ways. For example, one may use antibodies to any epitope of the target MHC antigen in conjunction with complement to kill any cells having the antigen. Alternatively, one may use conjugates of the appropriate antibody, particularly monoclonal antibody with a toxin, such as the A chain of ricin, abrin, diphtheria toxin, or the like. Affinity chromatography may be employed, where antibodies may be used to remove cells comprising the target antigen. The resulting cells which survive should be free of at least one MHC antigen on their surface and now not be as subject to transplant rejection when introduced in vivo as wild- type cells.
• the cells may then be grown in an appropriate nutrient medium for expansion and used in a variety of ways.
• the cells may be used for transplantation, to become part of an existing tissue, or may be grown to form tissue for transplantation into a non-syngeneic host.
• the cells may be used for replacement of skin in the case of burns, where keratinocytes may be grown to form a continuous layer prior to application.
• the keratinocytes may be used in the case of plastic surgery to replace skin removed from the host for use at another site.
• Other uses for the keratinocytes include transplantation in decubitus ulcers.
• islets of Langerhans may be grown and introduced into capsules or otherwise for insertion into a host for the production of insulin.
• retinal epithelial cells they may be injected into the subretinal space of the eye to treat visual disorders, such as macular degeneration.
• immune cells they may be injected into the bloodstream or elsewhere to treat immune deficiency.
• myoblasts they may be injected at various sites to treat muscle wasting diseases, such as Duchenne muscular dystrophy.
• non-syngeneic tissue such as xenogeneic grafts of heart or liver may be performed between related species.
• the cells may be employed as films, introduced in containers for maintenance at a particular site, or as solid masses impregnated in inert matrices or independent or a matrix.
• the number of cells administered will vary widely, depending upon the particular application and the manner in which the cells are administered. Administration may be by injection, topical application, incision and placement, in the appropriate location. Another situation is where one wishes to introduce a sequence at a target site which is refractory to homologous recombination.
• the subject methodology can be used to introduce a sequence of interest at a site proximal to a locus to be influenced by the sequence of interest, where the locus is refractory to homologous recombination, and one wishes to delete a region proximal to the refractory locus while bringing an exogenous sequence in proximity to the refractory locus, or the like.
• the target cells may be any of a variety of vertebrate cells, particularly animal cells, more particularly mammalian cells.
• embryonic stem cells which may be used to create transgenic animals, and thereby introduce the altered chromosome into the germline of a host animal.
• Embryonic cells of particular interest include rodent cells, e.g. mouse, rat and guinea pig.
• the method may be used to effect deletions in the chromosome of various sizes.
• Relatively small deletions 500 bp to 15 kb
• Such small deletions can also be achieved by standard targeting methodology, by choosing target regions for recombination that are separated by the desired deletion. If standard methodology is used, however, a positive marker must then be tolerated in the final locus. When no extra marker is desired in the final locus, then the method of this invention will permit one to effect a deletion without leaving behind any exogenous sequences. Large deletions, up to 4000 kb, can be achieved by the methodology of this invention.
• the size of deletions that can be made by standard methodology are restricted and vary in efficiency.
• the method of the invention is far more powerful than conventional techniques, because it relies, not on intermolecular homologous recombination for the excision step, but rather upon intrachromosomal recombination, using negative selection. Large deletion events resulting from the subject invention are more likely to occur at frequencies within the scope of a typical experiment, as compared to a deletion event resulting from a standard targeting experiment.
• deletion of large stretches of DNA may delete genes essential to the viability of the cell into which the targeting construct is introduced.
• the essential genes that may be lethal recessive genes, may be added back into the chromosome via the targeting construct or by secondary transfection with other vectors. The essential genes must be added back prior to rendering the deletion homozygous.
• the method of the subject invention may have a number of embodiments. Shown in Figure 2 is one embodiment of the invention, wherein a second negative selectable marker and ADH sequence are included in the construct. The second vADH will not normally share any sequence identity with the first v-ADH.
• the second c-ADH site will be located on the same chromosome as the first c-ADH and the c-target sequences, with the c-target sequences being located between the two c-ADH sequences.
• negative selection will be applied. This will select for those cells which have deleted the sequence between the first c-ADH and v-ADH.
• a second round of negative selection desirably for a different marker, will select for those cells which have deleted the sequence between the second v-ADH and c-ADH. In this way, the deletion will be bidirectional from the site of the initial targeting event.
• a series of v-ADH and negative selection marker sequences can be arranged in a sequential manner on the targeting construct to delete in successive stages chromosomal DNA sequences in one direction proceeding from the site of the initial targeting event on the chromosome ( Figure 3) .
• the targeting construct can be constructed with an arrangement of elements as described herein to accomplish bidirectional and staged successive unidirectional deletions to effect particular deletions in the chromosome.
• the ADH sequence may be a repetitive sequence.
• the end point for the deletion could be at a number of different sites, where ever the repetitive element is found.
• a series of nested deletions could be constructed, with a constant starting point for the deletion, and a variable amount of DNA deleted from that site.
• Repetitive sequences of interest include the Alu, LI, ⁇ -satellite, telomeric or sub-telomeric repeat sequences found in man, and analogous repeat sequences found in other mammals.
• the ADH sequence may be homologous to one of the target sequences, but incorporating a genetic lesion or modification.
• the genetic lesion or mutation may be an insertion, substitution or deletion in the DNA sequence.
• the number of altered nucleotides will usually be less than about 20 bp, more usually less than about 10 bp, and will be at least 1 bp. Chromosomal sequences will usually not intervene between the ADH and target sequences in this case, therefore the only sequences which are deleted will be vector sequences. In this way, a genetic modification can be introduced without leaving behind marker and vector sequences.
• the targeting vector was designed as a replacement type vector initially to delete the constant region as well as the J region of the kappa locus and replace it with three elements through homologous recombination using regions of homology flanking the constant region ( Figure 4) .
• a diphtheria toxin gene (A chain) flanking either or both regions of homology was included in some cases as a negative selectable marker.
• the three elements consisted of the G418 resistance drug marker, an ADH sequence of mouse DNA homologous to a region of the kappa locus located upstream of the J region, and a thymidine kinase gene.
• this initial targeting placed a second copy of the ADH sequence in the locus.
• This duplication was then used to effect a deletion of the sequences between the segments by applying selective pressure. In this case the cell deletes the thymidine kinase gene that lies between the two segments in order to survive gancyclovir selection.
• the regions of homology were derived from a 129 mouse fetal liver genomic library (Stratagene, San Diego, CA) which was screened using two probes. The first probe was a 1.6 kb Hpal/BamHI fragment (Steinmetz and Zachau, (1980), Nucleic Acids Research 8:1693-1706) that spans the constant region. A lambda phage clone which hybridized to this probe was identified and used to purify and isolate phage DNA.
• Phage DNA from a lambda clone positive for this probe showed that the probe hybridized to a 5.5 kb Sacl fragment which was subcloned into the Sad site of pBluescript SKI (Stratagene) to give the plasmid pSK.5'kappa ( Figure 5) .
• the inactivation vectors which contained a 5' region of homology, a thymidine kinase gene, an ADH, a neomycin resistance gene and a 3' region of homology ( Figure 6) flanked in some instances by diphtheria toxin genes were constructed from three plasmids ( Figure 5) containing: (a) the 5' fragment of homology with or without the diphtheria toxin gene (DT) driven by the mouse phosphoglycerate kinase gene (PGK) promoter as a negative selectable marker, (b) the herpes thymidine kinase gene (tk) driven by the mouse phosphoglycerate kinase gene (PGK) promoter as a negative selectable marker along with the ADH and the G418 selectable neomycin (neo) gene from pMClNeo (Thomas and Capecchi (1987), Cell 51:503-12), and (c) the 3' fragment of homology
• a diphtheria toxin gene cassette was created in which the gene was flanked by the PGK promoter and the bovine growth hormone polyadenylation signal (Woychik et al. (1984), Proc. Natl. Acad. Sci. U.S.A.. 81:3944-3948; Reifen et al. (1986), DNA 5:115-122) .
• a 2.3 kb Xbal/EcoRI fragment from pTH-1 Maxwell et aJ . (1986) , Cancer Res.
• hMTII human metallothionein
• a 0.5 kb Xbal/Pstl fragment from pKJl was joined to a 3.1 kb Xbal/Ncol fragment from pSK.DT using a Pstl/Ncol adapter formed from the oligonucleotides 5'-GGGAAGCCGCCGC-3' and 5'-CATGGCGGC GGCTTCCCTGCA-3 ' to give the plasmid pSK.pgkDT.
• a 248 bp fragment containing the bovine growth hormone polyadenylation signal obtained by PCR amplification of bovine genomic DNA using the oligonucleotide primers 5'-CAGGATCCAGCTGTGCCTTCTAGTTG-3' and 5'-CTGAGCTCTAGACC CATAGAGCCCACCGCA-3' , was cloned into pCRlOOO (Invitron Corp., San Diego, CA) . The polyadenylation sequence was then cloned behind the DT gene as a Hindlll/pvuII fragment into pSK.pgkDT cut with Hindlll and Hpal to give the plasmid pSK.pgkDTbovGH.
• the DT gene cassette from pSK.pgkDTbovGH was moved as a 2.1 kb EcoRI/Hindlll fragment into pSK.A cut with EcoRI and Notl using a Hindlll/Notl adapter formed from the oligonucleotides 5'-AGCTGGAACCCCTTGC-3 and 5'-GGCCGCAAGGGGTTCC-3' to give the plasmid pSK.A/DT.
• a Hindlll/Notl adapter formed from the oligonucleotides 5'-AGCTGGAACCCCTTGC-3 and 5'-GGCCGCAAGGGGTTCC-3'
• a 4.0 kb Sphl/Bsu361 fragment resulting from a partial Bsu36I digest followed by a complete Sphl digest of plasmid subclone pUC218/5.6kappa was ligated to pSK.A or pSK.A/DT to give the plasmids pSK.A/5'K and pSK.A/DT/5'K, respectively.
• the 5'-end of the DT gene and kappa fragment were adjacent to each other running in the opposite transcriptional orientations.
• the PGKtk gene from the plasmid pKJtk was cloned as a 2.7 kb EcoRI/Hindlll between the unique EcoRI and Hindlll sites of pSK.B to give pSK.B/TK.
• a 0.8 kb EcoRI fragment used for the ADH was cloned from pSK.5'kappa and was ligated into the EcoRI site of pSK.B/TK to give pSK.B/ (TK/0.8K) such that the 5'-end of the tk gene and kappa fragment were adjacent to each other running in opposite transcriptional orientations.
• the 1.1 kb neo gene from pMClNeo was cloned as an XhoI/BamHI fragment between the same sites of pSK.B/ (TK/0.8K) to give pSK.B/ (TK/O.8K/Neo) .
• the plasmid pSk.C/3'K containing the 3' fragment of homology was constructed by ligating pSK.C digested with BamHI and treated with alkaline phosphatase to the 1.1 kb Bglll/BamHI fragment isolated from pUC218/5.6kappa.
• pSK.C/3'K the kappa fragment was oriented such that transcription proceeded from the Sacl in the plasmid polylinker in the direction of the Kpnl site.
• the 2.1 kb DT cassette from pSK.pgkDTbovGH was cloned as an EcoRI/Hindlll fragment into the same sites of pSK.C to give pSK.C/3 , K/DT.
• the embryonic stem cell line E14-1 a subclone of E14 (Hooper et al., (1987) Nature 326:292-295), was cultured in DMEM with 4.5 g/1 glucose (JRH Biosciences, Irvine, CA) supplemented with 15% heat inactivated fetal calf serum, recombinant murine leukemia inhibitory factor (ESGRO from Gibco BRL, MD, 1000 units/ml), ⁇ -mercaptoethanol (0.1 mM) , glutamine (2 mM) and penicillin (100 U/ml) /streptomycin (0.1 mg/ml) and grown at 37°C in 5% C0 2 .
• ESGRO murine leukemia inhibitory factor
• the cells were cultured on mitomycin-treated primary embryonic fibroblast feeder layers essentially as described by Roller and Smithies, (1989) , supra.
• the embryonic fibroblasts were prepared from day 14 embryos carrying the homozygous targeted mutation of 3 2 -microglobulin (Roller et al., (1990) Science 248:1227-30). These feeder cells are capable of growth in media containing G418.
• the ES cells were prepared for electroporation by trypsinization, concentration by brief centrifugation and resuspension in HEPES-buffered saline at 2 X 10 7 cells/ml.
• the cells were equilibrated at room temperature, and DNA (20 ⁇ g) linearized (as described above) was added. The mixture was electroporated at 960 ⁇ F and 250 V with a BioRad Gene Pulser. The cells were left to stand at room temperature for 10 minutes before plating onto a 4 x 10 plate of mitomycin C treated feeders (3 x 10 6 feeder cells/plate) . After incubation at 37°C for 48 hr, the cells were fed media containing G418 at 150 ⁇ g/ml (effective concentration) .
• the individual surviving colonies were each picked and dissociated in a drop of trypsin in a 96-well plate and then incubated at 37°C for 2 min.
• the cells from each colony were transferred into a well of a 24-well plate containing mitomycin C treated feeder cells and selective media with G418 at 150 ⁇ g/ml. After an additional 5-8 days, 20% of the cells in each well were frozen and the remainder were used to prepare genomic DNA.
• the cells were lysed with 0.4 ml of 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM EDTA, 1% SDS, and proteinase R (1 mg/ml) by incubation overnight at 50°C.
• the DNA was purified by phenol extraction and ethanol precipitation. The DNA pellets were washed with 70% ethanol, dried and resuspended in TE (20 ⁇ l) .
• the fragment increased in size because the Bgl II site in the Bgl II/BamHI fragment was lost in the targeting plasmid due to the joining of a Bgl II site to a BamHI site in the ligation, and a new Bgl II site located in the thymidine kinase gene was introduced into the targeted locus.
• cells from clone 653 were plated on feeder cells at a density of 0.5-1 x 10 6 cells/10 cm dish in the presence of both gancyclovir (2 ⁇ M) and G418 (150 ⁇ g/ml) . After growth for 5 days in the presence of both drugs, clones were picked as described above into 24-well plates and grown under G418 selection alone. After an additional 5-8 days, 20% of the cells in each well were frozen and the remainder used to prepare genomic DNA as previously described.
• a Sacl digest of clone 653 DNA probed with the 0.8 kb EcoRI fragment showed a 5.5 kb fragment from both the unmodified and modified alleles and a 3.1 kb fragment from the targeted allele only ( Figure 7).
• the 5.5 kb fragment was also detected in DNA from clone 653B and an additional 2.0 kb fragment.
• the 5.8 kb Bgl II fragment and the 2.0 kb Sacl fragment were consistent with an analysis of the predicted restriction map for a precise excision step in which 10.3 kb of DNA were deleted including the J region, the tk gene, and one copy of the ADH.
• the unmodified E14-1 cells contributed to the germline at a high frequency after injection into C57BL/6J blastocysts.
• the ES cells (10-15) were injected into each blastocyst, and injected blastocysts (10) were transferred to a pseudopregnant female mouse, 5 into each uterine horn. Chimeric pups are identified by chimeric coat color. Chimeric males are bred to C57BL/6J females, and germline transmission of the 129 derived ES cell (unmodified or targeted) is detected by the agouti coat color of the FI offspring.

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• 1994
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