EP0608210A1 - Test de mutagenese a l'aide d'animaux transgeniques porteurs de sequences d'adn tests - Google Patents

Test de mutagenese a l'aide d'animaux transgeniques porteurs de sequences d'adn tests

Info

Publication number
EP0608210A1
EP0608210A1 EP91908707A EP91908707A EP0608210A1 EP 0608210 A1 EP0608210 A1 EP 0608210A1 EP 91908707 A EP91908707 A EP 91908707A EP 91908707 A EP91908707 A EP 91908707A EP 0608210 A1 EP0608210 A1 EP 0608210A1
Authority
EP
European Patent Office
Prior art keywords
dna sequence
test dna
genome
bacteriophage
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP91908707A
Other languages
German (de)
English (en)
Other versions
EP0608210A4 (fr
Inventor
Joseph A. Sorge
Jay M. Short
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Stratagene California
Original Assignee
Stratagene California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Stratagene California filed Critical Stratagene California
Publication of EP0608210A4 publication Critical patent/EP0608210A4/fr
Publication of EP0608210A1 publication Critical patent/EP0608210A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. 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
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • G01N33/5017—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity for testing neoplastic activity
    • 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
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • 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
    • A01K2267/0393—Animal model comprising a reporter system for screening tests
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Definitions

  • This invention relates to transgenic animals and to tests for monitoring mutagenic agents in live animals. More specifically, this invention relates to the creation of transgenic non-human animals carrying test DNA sequences and to methods for monitoring and assessing the mutagenic potential of agents by exposing the transgenic animal to one or more suspected mutagens, and optionally recovering the test DNA sequence, and examining the test DNA sequence for mutations. Novel methods for increasing the efficiency of test DNA sequence recovery and rapid analysis of specific test DNA mutations are also described.
  • Ames' test uses a special strain of bacteria to detect these mutations.
  • Ames, et al. An Improved Bacterial Test System for the Detection and Classification of Mutagens and Carcinogens, Proc. Nat. Acad. Sci.. 70:782-86 (1973).
  • This test and many analogues that use other types of bacterial or animal cells permit the rapid screening of very large numbers of cells for the appearance of an altered phenotype. The appearance of this altered phenotypic trait reflects the occurrence of a mutation within the test gene.
  • Eukaryotic cell lines have also been used to detect mutations.
  • Glazer et al. Detection and Analysis of UV-induced Mutations in Mammalian Cell DNA using Lambda Phage Shuttle Vector, Proc. Natl. Acad. Sci. USA. 83:1041-1044 (1986).
  • a target test gene the amber suppressor tyrosine tRNA gene of E. coli in a bacteriophage shuttle vector, was integrated into a genomic host mammalian cell line by DNA transfection of cultured cells in vitro. After exposing the host cell line to putative mutagenic agents, test genes were reisolated, propagated in bacteria, and analyzed for mutations.
  • the test is incapable of accurately monitoring mutagenic metabolites of the agent being tested that are only produced at the appropriate concentrations by differentiated cells or the tissue of live animals.
  • a two year study by the NIH concluded that data obtained from four different prokaryotic and eukaryotic in vitro assays had only a 60% concordance with whole animal carcinogenicity studies. Tennant et al.. Science, 236:933-941 (1987). The study suggests that the high rate of error may result from potential variation in genetic susceptibility between in vitro systems and whole animals.
  • metabolites frequently involved in activation of promutagens, are not present in in vitro systems, allowing mutagenic potential to go undetected.
  • differences in DNA repair mechanisms between prokaryotes and eukaryotes may account for some discrepancies in results.
  • Test genes and large scale screening assays used for in vitro assays are not available for live animal studies. Short of relying on long term animal studies that detect phenotypic changes that require a long time to be identifiable, such as tumors, organ failure, coat color, etc., current tests do not provide a means for monitoring organ-specific mutations of DNA. Hence, there exists a need for a system that places a test DNA sequence within an animal and is subsequently assayed on a large scale for mutations. There also exists a need for methods that detect mutations caused by chemical metabolites of the agent being tested. To be most effective the system needs to be capable of monitoring genetic changes in as many tissues of an animal and as easily, rapidly, and inexpensively as possible.
  • the present invention providing novel transgenic non-human mammals and methods utilizing such mammals for mutagenesis testing, satisfies these needs. More specifically, the present invention provides a sensitive screen for the mutagenicity of suspected agents and permits the monitoring of the mutagenic effects of such agents and the mutagenic effects of the metabolites of such agents. Additionally, the invention can permit the identification of the nature of the mutation, e.g., DNA transition, transversion, deletion, or a point or frameshift mutation. Further, the methods of the invention offer the significant advantage of being rapid to perform, thus permitting the identification of potential mutagens appreciably before other tests can be completed, and is inexpensive relative to other whole animal tests. And, the present invention substantially reduces the number of animals which must be used for mutagenesis testing.
  • the present invention contemplates a method for assaying the mutagenic potential of an agent.
  • the method comprises administering a predetermined amount of the agent to an animal containing cells having a genome characterized by the presence of an excisably- integrated lambda phage.
  • the phage contains a target gene operatively linked to prokaryotic expression signals.
  • the exposed animal is typically maintained alive for a predetermined period of time, usually from hours to months, and preferably from days to weeks, such as about 4 days to two weeks.
  • a predetermined amount of the excisably-integrated lambda phage is then rescued from cells harvested from the maintained animal.
  • the rescued lambda phage is then introduced into and expressed in a restriction system deficient microorganism.
  • the animal is a transgenic animal, such as a transgenic rodent, preferably a transgenic rat or mouse.
  • the cells are human cells present in a SCID mouse.
  • a preferred method has been developed which uses a target gene system comprised of a set of two genes, a test gene and a reporter gene, incorporated into animals or animal cells to screen for compounds having mutagenic, carcinogenic or teratogenic activity. Exposure of the animals or animal cells to compounds having any of these activities causes mutations resulting in alterations in expression of the reporter gene. Both the target gene and the reporter gene constructs are operatively linked to a prokaryotic promoter.
  • This method has several advantages over the prior art methods of screening for compounds having mutagenic or teratogenic activity.
  • the most significant advantage is the ease in detection and decrease in number of false positives.
  • the mutation of genes encoding reporter proteins has previously been used to assay for mutagenic activity, the mutational event resulted in the protein not being expressed. Detecting a single cell, or even a few cells, not expressing a protein, while surrounded by cells which express the protein, is difficult, tedious, and subject to a high percentage of error.
  • the mutational event ultimately results in the expression of a reporter molecule which would otherwise not be expressed, and which is readily detected.
  • animal cells will be used to include cells in cell cultures, embryos, and differentiated animals.
  • mutant will be used to include toxins, carcinogens, teratogens, and other agents which alter DNA or RNA sequence or expression, unless stated otherwise.
  • reporter genes are based on the following criteria: (i) the reporter gene product cannot be detrimental or lethal to the transformed cells, (ii) the gene product should provide a simple and sensitive detection system for its quantitation, and (ii) non-transformed cells should have a low constitutive background of gene products or activities that will be assayed.
  • Reporter genes which encode enzymes; antigens or other biologically active proteins which can be monitored easily by biochemical techniques are preferred. These include beta- galactosidase (Norton, P. A. and Coffin, J. M. , Mol. Cell. Biol. 5:281-290 (1985), peroxidase and luciferase (de Wet, J. R. et al., Mol. Cell. Biol. 7:725-737 (1987).
  • the test gene is selected from the group of sequences which encode regulatory molecules that bind to a sequence controlling reporter gene expression. These can be repressors or other regulatory molecules, including anti-sense RNA. In the most preferred embodiment, the lad repressor gene is used as a mutagenesis target. The inactivation of the repressor gene by a mutagenic even causes the transcription and translation of a defective repressor protein that is no longer able to repress expression of the reporter gene the lacz gene encoding beta-galactosidase. Alteration of the operator region for the reporter gene in a manner that prevents binding of the repressor protein produces the same effect. Derepression of the reporter gene can then be monitored by assaying for defined functions of the gene product.
  • the bacterial lac operator-repressor system is preferred because it is one of the most basic and thoroughly studies examples of a protein-nucleic acid interaction that regulates transcription of a gene, as described by Coulondre and Miller, Mol. Biol.. 117:577 (1977) and Miller, Ann. Rev. Genet.. 17:215 (1983).
  • This bacterial regulatory system has been transfected into mammalian cells and expression detected by addition of an inducer, isopropyl beta-D thiogalactoside (IPTG) , as reported by Hu and
  • laclq lac operator
  • the present invention contemplates engineered animal cells, animal embryos and differentiated animals having a genome characterized by the presence of an excisably-integrated lambda phage containing a target gene system.
  • the target gene system comprises a test gene (transcribable, and preferably translatable DNA sequence) .
  • the test gene is operatively linked to prokaryotic expression signals, such as a promoter, ribosome binding site, stop codon, and the like.
  • An rDNA of this invention contains a target gene system operatively link for prokaryotic expression to a lambda phage.
  • a target gene system typically is operatively linked to a lambda phage and can be comprised of any of a variety of test genes whose transcription results in a detectable phenotype or genotype where a mutation in the nucleotide sequence of a test gene measurably alters the detectable phenotype or genotype.
  • Typical test genes include genes that confer drug resistance or other selective advantage, or genes whose expression alters the expression of a second reporter gene. Exemplary drug resistance genes confer resistance to ampicillin, kanamycin, chloramphenicol and the like.
  • a preferred test gene is a repressor or activator gene product whose expression directly alters the expression of a reporter gene.
  • exemplary is the repressor protein encoded by the lad gene, and genetic variants of the lad gene that function to block transcription of the beta-galatosidase gene (lacZ) by binding to the operator region of the lacZ gene's expression signals.
  • lacZ is a reporter gene.
  • a preferred lad gene is the laclq variant that expresses eight- to ten-fold elevated levels of repressor protein and more tightly represses expression of the lacZ gene.
  • test gene of the target gene system is operatively linked to a reporter gene.
  • a reporter gene provides a means for detecting mutations in the test gene. Typical reporter genes are expressed when the test gene is not mutated, and are not expressed when the test gene is expressed. Thus a reporter gene is a gene that encodes a detectable phenotype or genotype and whose expression is under the control of the test gene.
  • Genes that encode detectable phenotypes include drug resistance markers, enzymes whose activity produces a detectable reaction product and the like.
  • a preferred reporter gene is the beta- galactosidase gene (lacZ) .
  • a preferred lacZ gene is one that utilizes alpha complementation, as described herein, whereby functional lacZ activity requires the association of the alpha portion of the lacZ gene product with the complementary portion of the lacZ referred to as the lacZ M15 gene product.
  • the test gene is operatively linked to prokaryotic expression signals to facilitate the rapid detection of mutations by the present invention.
  • the test gene system Upon excision rescue of a target gene system, the test gene system is introduced into a prokaryotic expression system, such as a bacterial cell lawn, so that dilutions of the test genes can be expressed and thereby observed (reported) to quantify the extent of test gene mutation.
  • test gene comprises a lad or laclq gene and includes a lacl promoter region.
  • a lambda phage of this invention comprises a target gene system that is excisably-integrated into the genome of an animal cell or embryo.
  • excisably- integrated is meant that the lambda phage comprises excision elements operatively linked to the genome that provide a means to conveniently remove the test gene system from the animal, cell or embryo genome subjected to mutagenesis conditions for the purpose of assessing the possible occurrence of mutation.
  • Exemplary excision elements are nucleotide sequences flanking the target gene, and if present the reporter gene, and other elements of the target gene system, that allow site-specific excision out of the genome to which the target gene system is operatively linked (integrated) .
  • Preferred nucleotide sequences are the cos sites, flp recombinase recognition sites, or loxP sites recognized by the Cre protein, all of whom are described more fully herein.
  • cos site excision elements because of the convenience and the efficiency of excision of the genes contained between the two cos site nucleotide sequences when utilizing lambda bacteriophage in vitro packaging extracts as described herein.
  • the excision elements of a target gene system confer the ability to readily recover the target gene system from the utagen exposure conditions to the prokaryotic expression medium in which the reporter gene is measured.
  • test gene system is the lambda LIZ alpha vector described herein in which a laclq test gene is operatively linked to the alpha- complementation-based lacZ alpha gene, where both test and reporter genes are under the control of prokaryotic expression signals, namely, lad promoter and lacZ promoter/operator sequences.
  • This preferred system further contains nucleotide sequences operatively linked to the test gene that define a prokaryotic origin of replication, a selectable marker (amp R ) and a filamentous phage origin of replication such that the test gene can readily be transformed into a "fl-type" nucleic acid sequencing vector for rapid determination of the nature of the mutation in the test gene.
  • This latter feature is provided according to the teachings of Short et al., Nucl. Acids Res.. 16:7583-7600 (1988), where the terminator and initiator domains of the fl intergenic region are separated and flank the test gene sequences of this invention to be recovered and sequenced.
  • a promoter is a sequence of nucleotides that forms an element of a structural gene transcriptional unit which controls the gene's expression by providing a site for RNA polymerase binding resulting in the initiation of the process of transcription whereby a gene is transcribed to form a messenger ribonucleic acid (mRNA) molecule.
  • An operator is a sequence of nucleotides that forms a site for specific repressor binding. Thus, operators are specific for a particular repressor.
  • a repressor binding site is considered specific if the equilibrium binding constant for repressor binding to the operator is greater than 10 "9 molar (M) , preferably greater than 10-10 M, and more preferably greater than 10 "11 M.
  • the equilibrium binding constant for a repressor binding to an operator can readily be measured by well known equilibrium dialysis methods, or in a nitrocellulose filter binding assay where repressor is immobilized on nitrocellulose and 32 P-labeled operator-containing DNA segment is presented in solution for binding to the immobilized repressor. See, Miller "Experiments in Molecular Genetics", p367-370. Cold Spring Harbor Laboratory, New York, 1972.
  • Operators function to control the promoter for a structural gene by a variety of mechanisms.
  • the operator can be positioned within a promoter such that the binding of the repressor covers the promoter's binding site for RNA polymerase, thereby precluding access of the RNA polymerase to the promoter binding site.
  • the operator can be positioned downstream from the promoter binding site, thereby blocking the movement of RNA polymerase down through the transcriptional unit.
  • Multiple operators can be positioned on a rDNA molecule to bind more than one repressor.
  • the advantage of multiple operators is several fold.
  • RNA polymerase binding or translocation down the gene can be effected.
  • the loop structure formed provides strong inhibition of RNA polymerase interaction with the promoter, if the promoter is present in the loop, and provides inhibition of translocation of RNA polymerase down the transcriptional unit if the loop is located downstream from the promoter.
  • a vector contemplated by the present invention includes a procaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a procaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
  • procaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
  • procaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant
  • Those vectors that include a procaryotic replicon may also include a procaryotic promoter capable of directing the expression (transcription and translation) of the gene transformed therewith.
  • a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention. Bacterial expression systems, and choice and use of vectors in those systems is described in detail in "Gene Expression Technology", Meth. Enzymol.. Vol 185, Goeddel, Ed., Academic Press, NY (1990).
  • Typical of such vector plas ids are pUC8, pUC9, pBR322 and pBR329 available from Bio-Rad Laboratories, (Richmond, CA) and pPL and pKK233-2, available from Pharmacia, (Piscataway, NJ) , or Clone Tech (Palo Alto, Ca) .
  • the present invention provides novel transgenic non-human animals and methods for monitoring the mutagenic effects of potential mutagenic agents.
  • at least one copy of at least one target test DNA sequence is introduced into cells of a non- human mammal thereafter bred to produce test animals.
  • substantially all of the cells will contain the test DNA sequence.
  • the test transgenic animal is then exposed to an agent suspected to be mutagenic and the test DNA sequence may be subsequently recovered from individual tissues of the transgenic animal.
  • the test DNA sequence may be transferred into a microorganism, although such recovery and transfer is not requisite, and assayed for mutations, allowing rapid examination of multiple tissue specific genetic mutations.
  • any animal suitable for mutagenic testing may be used as the starting organism.
  • single cell animal embryos are harvested, although there may be other cells facilitating the uptake and ultimate ubiquitous presence of the marker DNA in cells of a differentiated animal.
  • any number or variety of sequences coding for a phenotype or genotype that is detectable upon mutation may be used for introduction into the transgenic non-human mammals of the invention.
  • Vectors capable of facilitating the recovery of the test DNA sequence from the host mammal cells, and capable of allowing replication and expression of the sequence in a bacterial host are preferably used as carriers for the target test DNA sequence.
  • the construct for such a vector and insert preferably should contain regions for excision from the mammal host genome, and regions that allow replication in a bacterial host cell, as well as regions that permit expression and assay of the test DNA sequence. If integration into the host genome is not required, desired regions that allow for replication of the test DNA sequence in the animal host cells should be present. Elbrecht et al., Episomal Maintenance of a Bovine Papilloma Virus Vector in Transgenic Mice, Mol. Cell. Biol.. 7:1276- 1279 (1987) .
  • the test DNA sequence is introduced into the host mammal, preferably (but not necessarily) at the single-cell embryo stage, so as to provide the stable presence of the test sequence throughout cells of the differentiated animal.
  • the use of chimeric animals is also contemplated herein.
  • this involves the integration of the test DNA sequence into the mammal host genome, although methods that allow the test sequence to be stably and heritably present through the use of autonomously replicating vectors will also be useful.
  • Elbrecht et al. Episomal Maintenance of a Bovine Papilloma Virus Vector in Transgenic Mice. Mol. Cell. Biol.. 7:1276-1279 (1987) .
  • this may be accomplished using the techniques of microinjection, electroporation, dielectrophoresis or various chemically mediated transformation techniques, all of which are well known in the art.
  • other techniques may be necessary.
  • the transgenic cell or cells Following the introduction of the test DNA sequence and integration into the genome or cell, the transgenic cell or cells must be allowed to differentiate into a whole organism. This may be accomplished, for example, by embryo implantation into pseudopregnant females, or by other techniques allowing maturation of transgenic embryos. Once such maturation and differentiation has occurred, the animal is assayed for the presence of the test DNA sequence. Typically this involves removing small portions of tissue from the animal and using standard DNA hybridization assay techniques to detect the presence of the test DNA sequence. Transgenic animals carrying the test DNA sequence are thereafter bred and offspring carrying the test DNA sequence my be selected for mutagenesis testing. In accordance with the invention, the selected transgenic mammals are exposed to agents or substances in question under appropriate conditions. Such conditions will depend, for example, on the nature of the agent or substance, the purpose of the mutagenesis study and the type of data desired.
  • tissue may be removed from the test animal. Because in the preferred embodiment the test DNA sequence is present in essentially all tissues, the tissue type tested is not limited by the process of insertion of the test DNA sequence. Any desired tissue may be removed and assayed at the DNA, RNA, protein or substrate/product level, by various methods including, but not limited to, in situ hybridization to the DNA or RNA, PCR, protein or enzymatic assays (PCR Protocols. A Guide to Methods and Applications, eds. Innis, M. et al.. Academic Press, Inc., 1990; Maniatis et al. , Molecular Cloning. A Laboratory Manual. Cold Spring Harbor, New York 1982) .
  • genomic DNA may be purified from the tissue.
  • the target test DNA sequence which is integrated may then be rescued from the total genomic DNA of the host. This may be accomplished by excising it from the host genome or by suitable procedures allowing separation by size, weight or charge density. The method of rescue is dependent upon whether test DNA sequence is inserted into the genome, and whether flanking regions allow for excision, or whether the test DNA sequence is part of a replicating element allowing for separation techniques.
  • the rescued test DNA sequences may then be transferred into and expressed by microorganisms suitable for large scale screening techniques.
  • this involves excising the test DNA sequence vector from the genomic DNA by packaging the test DNA sequence with bacteriophage packaging techniques. This may require ligating the test DNA sequence into an appropriate vector or merely involve direct transformation into a microorganism.
  • Microorganisms containing the test DNA sequence vector are thereafter grown on indicator plates or in selective media. Those organisms have a phenotype indicating mutation of the test DNA sequence are considered to contain a mutated test DNA sequence.
  • Bacteriophage packaging techniques involve the use of bacteriophage-infected host cell extracts to supply the mixture of proteins and precursors required for encapsidating the bacteriophage DNA from exogenous sources.
  • rescue efficiency of the test DNA sequence can be significantly increased by eliminating the restriction systems in the strain of host microorganism used both for preparing the packaging extracts as well as those microorganisms used for plating to detect mutagenesis.
  • other recovery systems e.g., DNA transformation of isolated genomic DNA, would be improved by removal of such restriction systems or activities.
  • rescue efficiencies may be increased up to at least 12,000 pfu/ ⁇ g genomic DNA. These rescue efficiencies enable several million target genes from each tissue be analyzed, generating a large number of data points and resulting in a significant reduction in the numbers of animals required for mutagenesis testing with greater statistical significance.
  • the integrated target test DNA sequence is, preferably, rescued from the total genomic DNA of the host mammal using a lambda packaging extract deficient in restriction systems which recognize and deactivate foreign DNA.
  • the rescued test DNA sequences may then be transferred into and expressed by restriction system deficient microorganisms.
  • a shuttle vector system can be constructed which provides rapid analysis of test DNA sequence.
  • the test DNA sequence may be contained within a system which allows excision and recircularization of the test DNA sequence, which system is contained by a bacteriophage genome. Following rescue of the bacteriophage genome containing test DNA sequence using packaging extracts, the test DNA may be further excised from the bacteriophage genome and recircularized to provide for rapid mutation analysis.
  • the present invention contemplates the performance of mutagenesis testing by examining the phenotypes of cells containing the test DNA sequence without recovery of the test DNA sequence from the cell. This may be accomplished by the sectioning of tissues of the transgenic mammal of the invention, after exposure to a potential mutagenic agent, and assaying the genotype or phenotype of the test DNA sequence by in situ hybridization or, e.g., by staining of the tissue sections.
  • the present invention has application in the genetic transformation of multicellular eukaryotic organisms which undergo syngamy, i.e., sexual reproduction by union of gamate cells.
  • Preferred organisms include non-human mammals, birds, fish, gymnosperms and angiosperms.
  • the present invention contemplates a non-human mammal containing a modified lambda bacteriophage (rDNA) of the present invention excisably-integrated in the genome of the mammal's somatic and germ cells, i.e., a transgenic mammal.
  • Mammals containing a rDNA of the present invention are typically prepared using the standard transgenic technology described in Hogan et al., Manipulating the Mouse F ⁇ nT- ⁇ r ⁇ r>; A Laboratory Manual. Cold Spring Harbor, NY (1987); and Palmiter et al., Ann. Rev. Genet.. 20:465-499 (1986); which methods are described further herein.
  • transgenic mammals are also possible using the homologous recombination transgenic systems described by Capecchi, Science. 244:288-292 (1989) .
  • Preparation of transgenic mammals has also been described in U.S. Patent No. 4,736,866, No. 4,870,009, No. 4,873,191 and No. 4,873,316.
  • One technique for transgenically altering a mammal is to microinject a rDNA into the male pronucleus of the fertilized mammalian egg to cause one or more copies of the rDNA to be retained in the cells of the developing mammal.
  • Usually up to 40 percent of the mammals developing from the injected eggs contain at least 1 copy of the rDNA in their tissues.
  • transgenic mammals usually transmit the gene through the germ line to the next generation.
  • the progeny of the transgenically manipulated embryos may be tested for the presence of the construct by Southern blot analysis of a segment of tissue. Typically, a small part of the tail is used for this purpose.
  • the stable integration of the rDNA into the genome of the transgenic embryos allows permanent transgenic mammal lines carrying the rDNA to be established.
  • Alternative methods for producing a non-human mammal containing a rDNA of the present invention include infection of fertilized eggs, embryo-derived stem cells, totipotent embryonal carcinoma (Ec) cells, or early cleavage embryos with viral expression vectors containing the rDNA. See for example, Palmiter et al., Ann. Rev. Genet.. 20:465-499 (1986) and Capecchi, Science, 244:1288-1292 (1989).
  • a transgenic mammal can be any species of mammal, including agriculturally significant species, such as sheep, cow, lamb, horse and the like. Preferred are animals significant for scientific purposes, including but not limited to rabbits, primates and rodents, such as mice, rats and the like. A transgenic mammal is not human.
  • the present invention also contemplates a method of introducing a target gene system into a cell, i.e., genetically programming a cell within an organism by introducing a modified lambda genome containing a target gene system of the present invention into the genome of a zygote to produce a genetically altered zygote, or into the genome of individual somatic cells in the organism.
  • the genetically altered zygote is then maintained under appropriate biological conditions for a time period equal to a gestation period or a substantial portion of a gestation period that is sufficient for the genetically altered zygote to develop into a transgenic organism containing at least 1 copy of the rDNA.
  • genetically programming means to permanently alter the DNA content of a cell within an organism such as a mammal so that a prokaryotic target gene system has been introduced into the genome of the cells of the organism.
  • any multicellular eukaryotic organism which undergoes sexual reproduction by the union of gamete cells may be genetically programmed using an rDNA of the present invention.
  • multicellular eukaryotic organisms include amphibians, reptiles, birds, mammals, bony fishes, cartilaginous fishes, cyclostomes, arthropods, insects, mollusks, thallaphytes, embryophytes including gymnosperms and angiosperms.
  • the multicellular eukaryotic organism is a mammal, bird, fish, gymnosperm or an angiosperm.
  • a transgenic organism is an organism that has been transformed by the introduction of a recombinant nucleic acid molecule into its genome. Typically, the recombinant nucleic acid molecule will be present in all of the germ cells and somatic cells of the transgenic organism. Examples of transgenic organisms include transgenic mammals, transgenic fish, transgenic mice, transgenic rats and transgenic plants including monocots and dicots. See for example,
  • Methods for producing transgenic organisms containing a rDNA of the present invention include standard transgenic technology; infection of the zygote or organism by viruses including retroviruses; infection of a tissue with viruses and then reintroducing the tissue into an animal; and introduction of a rDNA into an embryonic stem cell of a mammal followed by appropriate manipulation of the embryonic stem cell to produce a transgenic animal.
  • Wagner, et al., U.S. Patent No. 4.873.191 Oct. 10. 1989
  • Rogers, et al. Meth. in Enzymol.. 153:253-277 (1987); Verma et al., PCT Publication No. W087/00551; Cocking et al., Science. 236:1259-1262 (1987); and Luskin et al., Neuron 1:635- 647 (1988) .
  • Transgenic mammals having at least 1 cell containing the rDNA's of a prokaryotic gene regulation system of the present invention can be produced using methods well known in the art. See for example,
  • transgenic mammal of the present invention is produced by:
  • a fertilized mammalian egg may be obtained from a suitable female mammal by inducing superovulation with gonadotropins.
  • gonadotropins typically, pregnant mare's serum is used to mimic the follicle- stimulating hormone (FSH) in combination with human chorionic gonadotropin (hCG) to mimic luteinizing hormone (LH) .
  • FSH follicle- stimulating hormone
  • hCG human chorionic gonadotropin
  • LH luteinizing hormone
  • the efficient induction of superovulation in mice depends as is well known on several variables including the age and weight of the females, the dose and timing of the gonadotropin administration, and the particular strain of mice used.
  • the number of superovulated eggs that become fertilized depends on the reproductive performance of the stud males. See, for example, Manipulating the Embryo: A Laboratory Manual. Hogan et al., eds.. Cold Spring Harbor, NY (1986).
  • the rDNA may be microinjected into the mammalian egg to produce a genetically altered mammalian egg using well known techniques.
  • the rDNA is microinjected directly into the pronuclei of the fertilized mouse eggs as has been described by Gordon et al., Proc. Natl. Acad. Sci.. USA. 77:7380- 7384 (1980). This leads to the stable chromosomal integration of the rDNA in approximately 10 to 40 percent of the surviving embryos. See for example,
  • rDNA is present in every cell of the transgenic animal, including all of the primordial germ cells.
  • the number of copies of the foreign rDNA that are retained in each cell can range from 1 to several hundred and does not appear to depend on the number of rDNA injected into the egg as is well known.
  • An alternative method for introducing genes into the mouse germ line is the infection of embryos with virus vectors. The embryos can be infected by either wild-type or recombinant viruses leading to the stable of integration of viral genomes into the host chromosomes.
  • Retroviral integration occurs through a precise mechanism, leading to the insertion of single copies of the virus on the host chromosome.
  • the frequency of obtaining transgenic animals by retroviral infection of embryos can be as high as that obtained by microinjection of the rDNA and appears to depend greatly on the titre of virus used. See, for example, van der Putten et al., Proc. Natl. Acad. Sci.. USA. 82:6148-6152 (1985).
  • Another method of transferring new genetic information into the mouse embryo involves the introduction of the rDNA into embryonic stem cells and then introducing the embryonic stem cells into the embryo.
  • the embryonic stem cells can be derived from normal blastocysts and these cells have been shown to colonize the germ line regularly and the somatic tissues when introduced into the embryo. See, for example, Bradley et al.. Nature. 309:255-256 (1984).
  • the embryo-derived stem cells are transfected with the rDNA and the embryo-derived stem cells further cultured for a time period sufficient to allow the rDNA to integrate into the genome of the cell. In some situations this integration may occur by homologous recombination with a gene that is present in the genome of the embryo-derived stem cell.
  • the embryo stem cells that have incorporated the rDNA into their genome may be selected and used to produce a purified genetically altered embryo derived stem cell population. See, for example, Mansour et al., Nature. 336:348 (1988).
  • the embryo derived stem cell is then injected into the blastocoel cavity of a preimplantation mouse embryo and the blastocyst is surgically transferred to the uterus of a foster mother where development is allowed to progress to term.
  • the resulting animal is chimeric in that it is composed from cells derived of both the donor embryo derived stem cells and the host blastocyst.
  • Heterozygous siblings are interbred to produce animals that are homozygous for the rDNA. See for example, Capecchi, Science. 244:1288-1292 (1989).
  • the genetically altered mammalian egg is implanted into host female mammals.
  • Methods for implanting genetically altered mammalian eggs into host females are well known. See, for example, Hogan et al. , Manipulating the Mouse Embryo: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1986) .
  • Pseudopregnant recipient females may be produced by mating females in natural estrus with vasectomized or genetically sterile males. After mating with a sterile male, the female reproduction tract becomes receptive for transferred embryos even though her own unfertilized eggs degenerate.
  • the genetically altered mammalian eggs are then transferred to the ampullae or the uterine horns of the pseudopregnant recipient. If the genetically altered mammalian egg is transferred into the ampullae it must be enclosed in a zona pellucida membrane. If it is transferred into the uterine horns the genetically altered mammalian egg does not require a zona pellucida membrane.
  • the host female mammals containing the implanted genetically altered mammalian eggs are maintained for a sufficient time period to give birth to a transgenic mammal having at least 1 cell containing a rDNA of the present invention that has developed from the genetically altered mammalian egg. Typically this gestation period is between 19. to 20 days depending on the particular mouse strain.
  • the breeding and care of mice is well known. See for example, Manipulating the Mouse Embrvo: A Laboratory Manual, Hogan et al., eds. , Cold Spring Harbor, New York, (1986) .
  • an animal that contains a target gene system in specific tissues or cells is used to test the effect of a material, composition, or compound suspected of being a carcinogen on the specific tissue.
  • the animal is exposed to the particular material or compound and the mutagenic effect on the animal is determined by the derepression of the operator-regulated reporter gene segment as an indication of the carcinogenicity of the compound or material.
  • composition suspected of having carcinogenic activity is introduced into the animal by any suitable method including injection, or ingestion or topical administration.
  • the animal is then maintained for a predetermined time period that is sufficient to allow the composition to produce a mutagenic effect on the genes of the target gene system.
  • this time period ranges from several minutes to several days depending on the time the composition requires to mutagenize the genes.
  • the physiological process or parameter assayed as an indication of mutagenesis depends upon the particular physiological alteration produced by the expression of the reporter gene.
  • a change in a physiologic parameter is determined by measuring that parameter before introduction of the composition into the animal and comparing that measured value to a measured value determined in identical manner after introduction of the composition into the animal.
  • the copy number of the test gene in the transgenic animals alters the sensitivity of transgenic animals to the effects of the suspected carcinogen. Therefore, selection of transgenic animals with varying transgene copy numbers of the test gene will alter the sensitivity of the transgenic mice to the suspected carcinogen.
  • BHB2690, which is D " are available from the American Type Culture
  • Step 1 A PI lysate is made from the E. coli K-12 strain described above.
  • Step 2 BHB2688 and BHB2690 are transduced with the PI lysate (Miller, J. , Experiments in Molecular Genetics- Cold Spring Harbor Lab. , Cold Spring Harbor, New York (1972)).
  • Step 3 Tetracycline (tet R ) resistant colonies are selected and purified.
  • Step 4 Loss of tetracycline resistance is selected for on Bochner plates (Bochner, B. R. , et al., J.
  • Step 5 Lack of mcrA restriction activity is tested by comparing transformation efficiency of unmethylated pBR322 versus pBR322 that has been in vitro methylated by Hpall methylase (Raleigh, supra) .
  • a mcrA+ strain will show a greatly reduced efficiency with the methylated plasmid. If mcrA activity is absent, this strain is then called BHB2688mcrA " and BHB2690mcrA * .
  • Step 8 Select for tet R colonies. Purify one colony that is also kan R .
  • Step 9. Select for loss of tet R on Bochner plates (Bochner, supra) .
  • Step 10 Purify several colonies and test for sensitivity to tetracycline and kanamycin. Select colonies that are both tet s and kan s .
  • Step 11 Test for lack of mcrB restriction activity as done for the mcrA test, however in this case, the pBR322 should be in vitro methylated by Alul methylase (Raleigh, supra; Ross, supra) . A mcrB+ strain will show a greatly reduced efficiency with the methylated plasmid.
  • Test for mrr restriction activity by comparing plating efficiency of lambda versus lambda which has been in vivo methylated by Pst I methylase (Heitman, supra) . An mrr+ strain will show reduced efficiency with the methylated lambda.
  • Test for hsdR restriction activity by comparing plating efficiency of lambda versus lambda which has been in vito methylated by hsdM methylase (Wood, W. , J. Mol. Biol.. 16:118-133, (1966); Adams, Bacteriophages. New York: Interscience 1959; Bickle, supra. at pp. 95- 100) .
  • hsdR+ strain will show reduced efficiency with unmethylated lambda. If a strain (purified colony) lacks all restriction activity mcrA, mcrB, mrr, hsdR and was constructed by this method, it should then contain a deletion throughout the mcrB region (AmcrB) . These strains are BHB2688R “ and BHB2690R " .
  • PI was preadsorbed by admixing 10 7 phage to 1 ml of the above admixture followed by maintenance at 20 minutes in a 30 degrees Celsius (30 ⁇ C) waterbath to form a phage-cell admixture.
  • the resultant agar-containing cell suspension was plated onto a freshly made LB plate which was maintained at 30 ⁇ C for 8 hours. At the end of the maintenance period, the soft agar layers was scraped into a small centrifuge tube.
  • E. coli lysogen BHB2690 (ATCC # 35132) was used as the specific strain for transduction.
  • E. coli BHB2690 which was RecA-, was first transformed with pJC859 to introduce a functional RecA protein into the lysogen.
  • pJC859 was a plasmid in which the nucleotide sequence encoding RecA had been inserted at the Bam HI site of the plasmid E. coli vector, pBR322 (ATCC # 31344) .
  • Maniatis et al. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, 2nd ed. ,
  • E. coli BHB2690 competent cells were prepared following standard procedures familiar to one skilled in the art. Maniatis et al., supra, Section 1.76. Alternatively, competent cells can be obtained commercially. Five ml of a fresh overnight culture of
  • BHB2690 recA+ to be transduced was resuspended in 5 ml of buffer consisting of 0.1 M MgS0 4 and .5 mM CaCl 2 according to the procedure by Miller. Miller, Experiments in Molecular Genetics.. Cold Spring Harbor Laboratory, 1972.
  • the BHB2690 recA+ cell suspension was then aerated by swirling at 30°C for 15 minutes. To each of 5 small test tubes, 0.1 ml of the aerated suspended cells was added. One hundred microliters (ul) of PI lysate, prepared above, was added to the first tube.
  • the PI lysate was serially diluted 10- fold for addition to the remaining tubes except for the last tube which did not receive PI and, thus, ser ed as a control.
  • a tube without cells containing PI w s also used as an additional control.
  • the Pi lysate was preabsorbed by maintaining the tubes at 30°C in a water bath for 20 minutes. Two hundred ul of 1 M sodium citrate was then added to each of the prepared tubes and the contents of each tube was then plated on tetracycline-containing plates to select for tetracycline resistant (tet R ) colonies.
  • tet R colonies were selected and purified following procedures well known to one skilled in the art. The tet R colonies were then replated on Bochner plates to select for the loss of tetR as described by Maloy. Maloy et al., J. Bacteriol.. 145:1110-1112 (1981).
  • tet- sensitive, tet s colonies were selected on a medium consisting of the following: 15 grams/liter (g/1) agar; 5 g/1 tryptone broth; 5 g/1 yeast extract; 4 milliliters/1 (ml/1) chlortetracycline hydrochloride (12.5 milligram (mg)/ml) ; 10 g/1 NaCl; 10 g/1 NaH 2 P0 4 - H 2 0; 6 ml/1 fusaric acid (2 mg/ml) ; and 5 g/1 ZnCl 2 (20 millimolar (mM)) . Chemicals were obtained from Sigma. (Sigma, St. Louis, MO) .
  • BHB2690RecA+mcrA- hereinafter designated BHB2690mcrA-, strains were, thus, determined and used to make BHB2690mcrB- transductions as described below.
  • BHB2690mcrB- The BHB2690mcrA- strains prepared above were used in similar transductions to select for BHB2690mcrB- strains.
  • a PI lysate was prepared as described above from any E. coli K12 strain that carried a TnlO (tet R ) in the mcrB gene (mcrB::TnlO (tet R ) .
  • the strain selected also carried the Tn5 with kanamycin (antibiotic) resistant gene (kan R ) in the mrr gene (mrr::Tn5 (kan R ) .
  • the E. coli BHB2690mcrA- (tet s ) strains were then transduced with PI lysate [(mcrB::TnlO (tet R ) and (mrr::Tn5 (kan R ) as described in Example la above. Tet R colonies that were also kan were selected and purified. The loss of tet R on Bochner plates was measured as described above. Colonies that were both tet s and kan s after selection on Bochner plates were purified.
  • the lack of mcrB restriction activity was performed as described for determining the lack of mcrA activity with the exception that pBR322 was in vitro methylated by Alu I methylase.
  • a mcrB+ strain showed a greatly reduced efficiency with the methylated plasmid.
  • the test for mrr restriction activity was accomplished by comparing plating efficiency of lambda versus in vivo methylated lambda (by Pst I methylase) .
  • a mrr+ strain showed reduced efficiency with the methylated lambda.
  • a separate test for hsdR restriction activity was also performed as the lack of activity confirmed the deletion of the entire mcrB region.
  • the hsdR restriction activity test was performed by comparing plating efficiencies of lambda versus lambda which had been in vivo methylated by hsdM methylase.
  • a hsdR+ strain showed reduced efficiency with the unmethylated lambda.
  • a selected colony which lacks all restriction activity, mcrA, mcrB, mrr, and hsdR, and constructed using this transduction approach was shown to contain a deletion throughout the mcrB region.
  • This strain was designated BHB2690R- and used in the preparation of extract for prehead as described in Example 2a.
  • B. E. coli BHB2688R- E. coli BHB2688 strains containing RecA+ but lacking mcrA, mcrB, mrr and hsdR were prepared using the approach described above for preparing E. coli BHB2690R-.
  • E. coli lysogen BHB2688 ATCC # 35131 was used.
  • the resultant strain, designated BHB2688R- was used in the preparation of extract for protein donor a described in Example 2 below.
  • coli BHB2690R- is picked and maintained overnight at 32 ⁇ C and 45 ⁇ C.
  • the bacteria with the mutation only grow at 32 ⁇ C and grow slowly due to the recA- mutation present in the BHB strains.
  • a 100 ml subculture of the verified master stock of E. coli strain BHB2690R- is then prepared and maintained overnight at 32 ⁇ C.
  • the optical density (OD) is measured at a wavelength of 600 nm.
  • NZM broth is prepared by admixing 10 g NZ amine, 5 g NaCl, and 2 g MgS ⁇ 4 -7H 2 0 to 950 ml of deionized water; the pH of the solution containing dissolved solutes is adjusted to pH 7.0 with 5 N NaOH) , prewarmed to 32 ⁇ C, in a 2-liter flask, to result in a starting of approximately 0.1.
  • the bacterial admixture is then maintained at 32 ⁇ C with vigorous agitation (300 cycles/minute in a rotary shaker) until an OD ⁇ o of approximately 0.3 is reached.
  • the OD ⁇ o of 0.3 is generally attained within 2 to three hours of maintaining the culture.
  • the cultures must be in the mid-log phase of growth prior to induction as described below.
  • the lysogen is induced by placing the flask in a water bath preheated to 45 ⁇ C. The flask is swirled continuously for 15 minutes.
  • An alternative approach for inducing lysogen is to immerse the flask in a shaking water bath set at 65 ⁇ C. The temperature of the fluid contents of the flask is monitored. When the fluid reaches 45 ⁇ C, the flask is then transferred to a water bath set at 45°C and maintained for 15 minutes. The induced cells are then maintained for 2 to 3 hours at 38 to 39 ⁇ C with vigorous agitation as described above. A successful induction is verified by the visual clearance of an added drop of chloroform to the culture.
  • the cells are recovered from the admixture by centrifugation at 4000g for 10 minutes at 4°C.
  • the resultant supernatant is decanted and any remaining liquid is removed with a pasteur pipette and a cotton swab.
  • the walls of the centrifuge bottle are wiped dry with towels.
  • Sonication buffer consists of 20 mM Tris- HC1, pH 8.0, (Tris[hydroxymethyl]-aminomethane hydrochloride) , 1 mM EDTA, pH 8.0, (ethylenediaminetetraacetic acid) and 5 mM beta- mercaptoethanol.
  • the bacterial cell pellet is resuspended in the sonication buffer by mixing to form a homogenized cell suspension.
  • the resultant suspension is transferred to a small, clear plastic tube (Falcon 2054 or 2057, Falcon, Oxnard, California) for subsequent sonication.
  • the cells are disrupted by sonication with 10 second bursts at maximum power using a microtip probe.
  • the tube containing the suspension is immersed in ice water and the temperature of the sonication buffer should not be allowed to exceed 4°C.
  • the sample is cooled for 30 seconds in between each sonication burst.
  • the sonication procedure is continued until the solution in the tube clears and its viscosity decreases.
  • the sonicated bacterial sample is transferred to a centrifuge tube and debris is pelleted by centrifugation at 12,000g for 10 minutes at 4"C forming a clear supernatant.
  • the resultant supernatant containing preheads is removed and admixed with an equal volume of cold sonication buffer and one-sixth volume of freshly prepared packaging buffer to form a diluted prehead admixture.
  • Packaging buffer consists of the following: 6 mM Tris-HCl, pH 8.0; 50 mM spermidine; 50 mM putrescine; 20 mM MgCl 2 ; 30 mM ATP, pH 7.0; and 30 mM beta-mercaptoethanol.
  • the admixture is then dispensed into pre-cooled to 4'C 1.5-ml microfuge tube in 15 ul aliquots.
  • the caps of the microfuge tubes are then closed and the tubes are immersed briefly in liquid nitrogen for freezing.
  • the frozen preheads in packaging buffer are then stored at -70°C for long- term storage.
  • the pelleted cells are resuspended in a total of 3 ml of ice-cold sucrose solution (10% sucrose in 50 mM Tris-HCl, pH 8.0) to form a cell suspension.
  • the resultant cell suspension is dispensed in 0.5 ml aliquots into each of six precooled to 4°C microfuge tubes.
  • Twenty-five ul of fresh, ice-cold lysozyme solution (2 mg/ml lysozyme in 10 mM Tris-HCl, pH 8.0) is admixed to each tube containing the cell suspension.
  • the cell-lysozyme admixture is gently mixed to form an E. coli extract and then immersed in liquid nitrogen for freezing.
  • the frozen tubes are removed from the liquid nitrogen and the extracts are thawed on ice.
  • Twenty- five ul of packaging buffer, as prepared above, is admixed to each tube containing thawed extract to form a packaging buffer-extract admixture.
  • the separately prepared admixtures are then combined in a centrifuge tube and centrifuged at 45,000g for 1 hour at 4°C to form an supernatant containing packaging protein donor.
  • the resultant supernatant is removed and dispensed in 10 ul aliquots into precooled at 4°C microfuge tubes.
  • the caps of the tubes are closed and the tubes are then immersed in liquid nitrogen.
  • the tubes are then removed from the liquid nitrogen and stored at -70°C for long term storage.
  • the frozen tubes containing prehead and packaging donor extracts prepared in Example 2a and 2b, respectively, are removed from storage at -70°C and allowed to thaw on ice.
  • the frozen-thawed lysate containing the protein donor thaws first and is admixed to the still-frozen sonicated prehead extract to form a prehead-protein donor admixture.
  • the resultant admixture is mixed gently until almost totally thawed.
  • the DNA to be packaged (up to 1 ug dissolved in 5 ul of 10 mM Tris-HCl, pH 8.0, 10 mM MgCl 2 ) is admixed with the thawed combined extracts and mixed with a fine glass stirring rod to form a DNA-extract admixture. The admixture is then maintained for 1 hour at room temperature.
  • SM buffer is prepared by admixing 5.8 g NaCl, 2g MgS0 4 -7H 2 0, 50 ml Tris-HCl, pH 7.5, and 5 ml 2% gelatin (w/v) to 1 liter of deionized water and adjusting the pH to 7.5) and a drop of chloroform is added and gently mixed. Debris is removed by centrifugation at 12,000g for 30 seconds at room temperature in a microfuge. The resultant supernatant is removed and contains packaged bacteriophage DNA particles. The titer of the viable bacteriophage particles is measured by plating on the appropriate indicator strains.
  • Recombinant DNAs that are 90% or 80% of wild-type bacteriophage lambda in length are packaged with efficiencies that are 20-fold to 50-fold lower, respectively, than those obtained with unit- length bacteriophage lambda.
  • the same packaging extracts may be used for the packaging of both bacteriophage lambda and cosmids.
  • the test sequence DNA can, theoretically, contain any number or variety of genes or other identifiable test DNA sequences.
  • an E. coli bacteriophage lambda genome has been engineered to carry lacZ, a beta- galactosidase test DNA sequence.
  • Lambda shuttle vectors L2B (46.5kb) or C2B (48.0kb) may be used.
  • the genotype of the modified lambda genome L2B is Lac5 delta (shind III lambda 2-3°) srl lambda 3-5° cI857 sXhL lambda 1" sScII lambda 4°.
  • this lambda DNA was diluted to a concentration of 10 micrograms per milliliter and the cos ends were annealed and ligated under conditions predominantly forming circular lambda phage monomers.
  • Maniatis et al. Molecular Cloning. A Laboratory Manual, pp. 109-110, 383-389 (Cold Spring Harbor, New York 1982) .
  • a variation of L2B may be constructed that contains a plasmid sequence that can be readily excised from the lambda phage and contains the lad repressor gene. This variation has several advantages.
  • DNA as described above was loaded in the injection pipet at a concentration of 1 to 10 micrograms per milliliter. Approximately one picoliter, as judged by a refractile change (Hogan et al. , supra) of the pronucleus, of DNA solution was injected into the male pronucleus. After DNA injection, four hundred embryos were transferred to Ml6 medium and incubated at 37°C in a 5 % C ⁇ 2 atmosphere for one to two hours. One hundred fifty embryos survived microinjection. Lysed embryos were discarded and 30 embryos that appeared normal were transferred to one of the fallopian tubes of each of 5 pseudopregnant foster mothers. The transfers were performed under a dissecting microscope using general anesthesia (avertin) .
  • mice Seven pups were born. After birth, newborn mice were kept with their foster mothers for 2 weeks, at which point they were then weaned and screened for DNA integration. A 2 cm portion of the tail was removed and homogenized in 2 ml of a solution of 0.1 M NaCl, 50 mM Tris-HCl, pH 7.5, lmM EDTA for short duration, but long enough to disrupt cell and nuclear membranes. The homogenized tissue was treated with 50 U/ml RNaseA and 0.1% SDS for 15 minutes at 37°C. The mixture was exposed to Proteinase K digestion for 3 hours at 55 ⁇ C followed by three extractions with phenol/chloroform. DNA was then precipitated by the addition of ethanol.
  • the filter with transferred DNA was hybridized overnight with 32 P labeled lambda DNA prepared according to standard procedures by the method of nick translation. Maniatis, supra. Following this overnight hybridization, the filter was washed in 0.1 x SSC, 0.1% SDS at 50"C and Kodak XAR film was exposed to it in order to identify lambda DNA present within the mouse genome. Lambda DNA, used as standards, that had been electrophoresed alongside the mouse genomic
  • transgenic mouse DNA were compared in intensity to the transgenic mouse DNA hybridized to the 3 P labeled lambda DNA to estimate the number of copies of test DNA per mouse cell. Three transgenic animals have been produced and identified by this technique.
  • mice tested for the presence of the test DNA sequence by the tail-blotting procedure (Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, pp. 174-183, Cold Spring Harbor Laboratory, 1986) were found to carry the test DNA sequence in DNA isolated from their tails. Eight weeks after birth these transgenic mice were mated and their progeny were examined for the test DNA sequence. Approximately 50% of the resulting offspring carried the test DNA sequence, demonstrating that the original transgenic mice carried the test DNA sequence in their germ line and that this sequence was inherited normally. While transgenic lines having approximately one copy of the test DNA sequence per cell can be obtained, it will be understood by one skilled in the art that multiple copy numbers per cell are obtainable and may be useful for many different applications.
  • rescue of the marker DNA sequence was accomplished by containing it within a lambda bacteriophage genome.
  • the entire lambda bacteriophage genome is excised from the mouse chromosome by the in vitro packaging extract.
  • the packaging extract recognizes the cos sites of the integrated lambda DNA and packages the sequences between the cos sites into lambda phage particles, as shown in Figure 1.
  • the test DNA sequence may be found within the genomic DNA purified from any tissue of the transgenic mouse. Since the test DNA sequence is contained within a lambda phage genome, it can be excised away from the remainder of genomic DNA by using a lambda phage packaging extract.
  • Packaged lambda phage such as L2B or C2B, may then be plated on E. coli cells for further evaluation.
  • Bacteriophage lambda DNA can be packaged in vitro using protein extracts prepared from bacteria infected with lambda phage lacking one or more genes for producing the proteins required for assembly of infectious phage particles.
  • Typical in vitro packaging reactions are routinely capable of achieving efficiencies of 10 8 plague from units (pfu) per ⁇ g of intact bacteriophage lambda DNA.
  • About 0.05 - 0.5 percent of the DNA molecules present in the reaction can be package into infectious virions.
  • the E protein is the major component of the bacteriophage head and is required for the assembly of the earliest identifiable precursor.
  • Bacteriophages mutant in the E gene accumulate all of the components of the viral capsid.
  • the D protein is localized on the outside of the bacteriophage head and is involved in the coupled process of insertion of bacteriophage lambda DNA into the "prehead” precursor and the subsequent maturation of the head.
  • Bacteriophages mutant in the D gene (D * ) accumulate the immature prehead but do not allow insertion of bacteriophage lambda DNA into the head.
  • the A protein is involved in the insertion of bacteriophage lambda DNA into the bacteriophage prehead and cleavage of the concatenated precursor DNA at the cos sites.
  • Bacteriophages mutant in the A gene also accumulate empty preheads. Complementing extracts have been prepared from cells infected with A " and E " or D " and E " strains; alternatively, extracts prepared from cells infected with A " mutants can be complemented by the addition of purified wild-type A protein.
  • a bacteriophage lambda DNA packaging extract is a proteinaceous composition that is capable of packaging bacteriophage lambda DNA into infectious virus particles.
  • the lambda DNA packaging extracts useful in this invention have a packaging efficiency of at least 10 8 , and more preferably at least 10 9 , pfu/ ⁇ g of intact lambda DNA.
  • the packaging extracts of this invention are usually prepared from cells containing bacteriophage lambda lysogens of the appropriate genotype, e.g., amber mutations in genes A, D, E and the like.
  • useful lysogens preferably have one or more of the following mutations: clts857 - specifies a temperature-sensitive bacteriophage lambda repressor molecule. This mutation causes lambda DNA to be maintained in the lysogenic state when the host bacteria are grown at 32 ⁇ C; bacteriophage growth is induced by transiently raising the temperature to 42-45 " C to inactivate the repressor specified by the cl gene.
  • Sam7 - an amber mutation in the bacteriophage
  • This mutation causes capsid components to accumulate within SuIII " bacterial cells for 2-3 hours following induction of the clts857 lysogen.
  • b-region deletion (b2 or bl007) - a deletion in the bacteriophage genome that effectively removes the lambda DNA attachment site (att) .
  • This mutation reduces, but does not entirely eliminate, the packaging of endogenous lambda DNA molecules in extracts made from the induced cells. red3 (in lambda) and recA (in E.
  • a lambda lysogen useful for producing a packaging extract is one deficient in one or more of the mcrA, mcrB, hsd and mrr restriction systems.
  • the genes comprising these systems can be removed or inactivated by well known methods, such as by transduction, transposon (Tn) mutagenesis, and the like.
  • Packaging extracts are usually prepared from a lysogenic bacteria having one or more of the following mutations: mcrA “ , mcrB “ , mrr “ , hsdR “ , and preferably from K-12 AmcrB, BHB2690R “ , or BHB2668R “ , by growing the appropriate lysogenic bacteria to mid-log phase at 32 ⁇ C, inducing lytic functions by inactivating the cl repressor protein by raising the temperature to 45 ⁇ C for 15 minutes, and then growing the cultures for an additional 2-3 hours at 38-39 ⁇ C to allow packaging components to accumulate. Cell extracts are then prepared as described further herein.
  • the plates are incubated overnight at 37 ⁇ C.
  • the phage plaques turn blue if the beta- galactosidase sequence within the lambda genome has not mutated.
  • a white plaque or faint blue plaque on the petri dish is evidence that a mutation in the beta-galactosidase sequence has, for example, altered the reading frame, altered essential codons, or has created stop codons in the sequence.
  • These white or faint blue plaques will be scored as positive for mutations and they can be plaque purified and saved for further analysis.
  • the ratio of white or faint blue to blue plaques minus background mutation rate from non-mutagenesis potency of the agent being tested when compared with DNA extracted from mice that have not been treated with potentially mutagenic agents.
  • test DNA sequence rescue efficiency can be influenced by the state of CpG methylation in the mouse chromosome. Highly methylated DNA may not be efficiently excised by lambda packaging extract, presumably because of inhibition of cleavage at the cos sites, inhibition of expression of lambda genes encoded on lambda phage, or restriction by E. coli restriction systems. This may be alleviated by placing transcriptional enhancers, promoters and/or other regions of the DNA which inhibit methylation near critical sites such as the cos site to reduce CpG methylation.
  • the drug 5'- azacytidine can also be used to reduce the level of DNA methylation in the target cells prior to DNA purification and rescue. Jaenisch, R.
  • fibroblast cell lines are obtained from organisms containing the test DNA sequence of interest. Adams, R.L.P., Cell Culture for Biochemists. pages 68-83 (1980) Elselvier/North Hollan Biomedical Press) . The cells are exposed in vitro at 37 ⁇ C, within 50 ⁇ M 5'azacytidine supplementing the culture medium. Upon DNA replication, the daughter DNA loses its CpG methylation, which eliminates the methylation of sites in the target vector, where the target vector is a lambda phage. The DNA from these fibroblasts is then exposed to in vitro packaging extract, as previously described.
  • organisms containing the test DNA sequence can be directly injected with a 1 mg/ml solution of 5'-azacytidine in 0.15 M NaCl. This is done over a period of at least about 4 days, with a total of 400 ⁇ g administered. Jaenisch, supra. After this treatment, DNA can be extracted from various tissues and packaged as before.
  • Plating Strain Restriction Systems We have determined that the efficiency of test DNA sequence recovery is dependent on the genotype of both the bacterial strain used to generate the packaging extract as well as the plating strains used for mutagenesis testing. This is due to host- controlled restriction systems that enable a bacterial cell to identify and inactivate foreign DNA by endonuclease cleavage. DNA is susceptible to restriction by the endonucleic activity of the host unless it is protected by modifications, such as methylation of specific nucleotides. While methylation of specific nucleotides usually serves to protect DNA from restriction by the endonucleolytic activity of the host, methylation at some DNA sequences actually confers sensitivity to restriction. One example, the mcrB restriction system of E.
  • coli K- 12 is responsible for the biological inactivation of foreign DNA that contains 5-methylcytosine residues. Ross et al.. Journal of Bacteriology , 171:1974-1981 (1989) .
  • restriction/methylation systems endogenous to E. coli which are capable of inactivating foreign DNa by endonuclease cleavage.
  • the most widely known systems are hsd (Bickle, T. Nucleases. p. 85, Cold Spring Harbor Lab., Cold Spring Harbor, N.Y. 1982), mrr (Heitman, J. et al., J.
  • the mrr system also involves adenine methylation, however, in this case the methylation does not serve to protect the DNA, but serves to make the DNA vulnerable to the restriction system.
  • the systems mcrA and mcrB are similar to mrr in that they recognize and restrict methylated DNA. However, these two systems differ from mrr in that they recognize methylated cytosine. Further, the mcrB function is provided by the products of at least two genes, mcrB and mcrC (Ross et al., _ _ Bacteriol.. 171:1974-1981 (1989)).
  • E. coli restriction systems In addition, naturally occurring strains of E. coli that are deficient in these restriction systems may be isolated and used. Identification of the genes responsible for the E. coli restriction systems was achieved by examination of the inhibitory effect of certain E. coli strains on the ability to recover lambda phage. Isolation of the responsible genes was achieved through the use of interrupted matings and PI transduction. An approximately 200 kb region of DNA in E. coli K-12 was found to produce an inhibitory effect on the plating efficiency of the rescued vector. Further, the region responsible for decreasing rescue efficiency was found to be near 98 minutes in the E. coli K-12 genetic map (Bachmann, B. E. coli and S. Typhimurium: Cellular and Molecular Biology. eds.
  • Strain RR1-A and K-12AmcrB are constructed as described below.
  • Step 1 A PI lysate is made from the E. coli K-12 strain described above.
  • Step 3 Tetracycline resistant colonies are selected and purified.
  • Step 4 Loss of tetracycline resistance is selected for on Bochner plates (Bochner, B.R. , et al., J. Bacteriol.. 143:926- 933 (1980)), and colonies are purified.
  • Step 5 Lack of mcrA restriction activity is tested by comparing transformation efficiency of unmethylated pBR322 versus pBR322 that has been in vitro methylated by Hpall methylase (Raleigh, supra) . A mcrA+ strain will show a greatly reduced efficiency with the methylated plasmid. If mcrA activity is absent, this strain is then called RR1-A.
  • Strain K-12AmcrB is constructed using two donor E. coli K-12 strains with the relevant genotypes mcrB: :TnlO(tet R ) , mrr: :Tn5(Kan R ) and mcrA: :TnlO(tet R ) and a recipient E. coli K-12 with the relevant genotype recA+, tet s .
  • Steps 1-5 Perform steps 1-5 as described for construction of RR1-A. In step 2, transduce any E. coli K-12 recA+ strain.
  • Step 6 Make a PI lysate from an E.
  • Step 8 Select the tet R colonies. Purify one colony that is also kan R .
  • Step 9 Select for loss of tet R on Bochner plates (Bochner, supra) .
  • Step 10 Purify several colonies and test for sensitivity to tetracycline and kanamycin. Select colonies that are both tet s and kan s .
  • Step 11 Test for lack of mcrB restriction activity as done for the mcrA tet, however in this case, the pBR322 should be in vitro methylated by Alul methylase (Raleigh, supra; Ross, supra) . A mcrB-i- strain will show a greatly reduced efficiency with the methylated plasmid. Test for mrr restriction activity by comparing plating efficiency of lambda versus lambda which has been in vivo methylated by Pst I methylase (Heitman, supra) . An mrr+ strain will show reduced efficiency with the methylated lambda.
  • hsdR-i- strain will show reduced efficiency with unmethylated lambda. If a strain (purified colony) lacks all restriction activities, namely, mcrA, mcrB, mrr, hsdR and was constructed by this method, it should then contain a deletion throughout the mcrB region (*mcrB) . It will then also very efficiently plate lambda that has been rescued from the mouse. This strain is called K- 12 mcrB.
  • the "A" symbol in Table 1 indicates that the strain contains a large deletion in the mcrB region. All other mcrB- strains listed in TAble l are K-12 derivatives believed to contain a small mutation in the mcrB region, with the exception of E. coli C which does not contain the K-12 mcrB region, and RRl-A which carries the wild type mcrB locus of E. coli B. It is known that all of these strains plate control L2B phage (amplified in hsdM+ E. coli K-12 rather than rescued from the mouse) with equal efficiency (within 1-4 fold) .
  • E. coli K-12 LCK8 was cloned.
  • a partial LCK8 genomic library was made in pOU61cos. (Knott, V. et al., Nucleic Acid Res.. 16:2601-2612 (1988)), packaged with GigapackTM II XL (Stratagene, La Jolla, Calif.), and plated on E. coli C.
  • Clones containing the 98 minute region were identified by colony hybridization using an oligonucleotide (ATGAGTGCGGGGAAATTG) probe specific to the hsd region (Gough, J.
  • SCS-8 Preferred E. coli strains for rescue of the lacZ construct from the transgenic animal genome are SCS-8 (Catalog Number 200,288) and VCS257 (Catalog Number 200,256) which are commercially available from Stratagene Cloning Systems and are also contained in a ki ⁇ t (Bi•g BlueTM Mouse Mutagenesi»s System, Catalog Number 720,000).
  • SCS-8 has the following genotype: recAl, endAl, mcrA, (mcrBC-hsdRMS-mrr) , A(argF- lac)U169, phi80AlacZAM15, Tnl0(tet r ).
  • SCS-8 provides the lacZAMIS gene which allows for alpha- complementation when SCS-8 is infected by the packaged bacteriophage.
  • Additional commercially available E. coli strains which contain the lacZAM15 genotype for use in this invention include the following: XLl-Blue (Stratagene, Catalog Number 200,236); SureTM (Stratagene, Catalog Number 200,238); PLK-F' (Stratagene, Catalog Number 200,237); JM101 (Stratagene, Catalog Number 200,234) ; JM109 (Stratagene, Catalog Number 200,235); and NM522 (Stratagene, Catalog Number 200,233).
  • mcrB deletion strains is described herein for use in mutagenesis testing and recovery of lambda phage DNA from mammalian cells, it is apparent that restriction system deficient strains may be used for other eukaryotic DNA cloning projects.
  • restriction system deficient strains may be used for other eukaryotic DNA cloning projects.
  • any number or variety of test DNA sequences or genes can be inserted between lambda cos sites.
  • the in vitro packaging extract would still excise the DNA between the cos sites and package it into a lambda phage particle.
  • a variety of recombinant lambda genomes or cosmids may be used for this excision event.
  • Mutations evidenced by the production of white plaques resulting from disruption of the ⁇ - galactosidase (0-gal) gene are useful for determining the mutation rate of a mutagen, but give little information regarding the specific mutation within the DNA.
  • analysis of the specific mutation is hampered somewhat by the size of the test j8-gal gene (i.e., about 3200 b.p.).
  • the target lambda phage can be made to provide a target gene with reduced size (e.g., the lad gene having about 1000 b.p.), and a rapid means with which the target gene is transferred from the lambda phage into plasmid vectors for sequence analysis.
  • both the lad and /9-gal genes are inserted within a lambda vector, such that if the mutation occurs within the lad gene, the repressor activity is lost allowing the 3-gal gene to be expressed giving rise to blue plaques in the absence of IPTG.
  • the lad gene is positioned upstream of the alpha portion of the lacZ gene in the vector (Miller, J.H. and Reznikoff, W.S., The Operon. 2nd Ed. Cold Spring Harbor Laboratory, 1980, pp. 104- 105) .
  • lacZAM15 complementary portion of the lacZ gene
  • the gene products synthesized by these two partial genes combine to form a functional 0-galactosidase protein (referred to as alpha-complementation) giving rise to blue plaques in the presence of Xgal when a mutation has occurred in the lad gene or in the presence Xgal and IPTG when the lad gene is not mutated.
  • alpha-complementation a functional 0-galactosidase protein
  • the AMIS portion of the lacZ gene provided by the host is provided either episomally (via a low copy number plasmid or F-factor) or stably integrated into the bacterial chromosome.
  • lacZ The alpha portion of lacZ is used because 1) the ⁇ -gal protein formed by alpha- complementation is known to be weaker in activity than the contiguous protein, minimizing the possibility of background blue plaques due to inefficient repression by lad, and 2) to provide a smaller and thus more easily characterized lacZ target should this gene be used in mutagenesis studies.
  • the requirements of the host E. coli in this system are the following: lacl(-), lacZAMl ⁇ , restriction(-) . All cloning steps are outlined in the figures 4 through 8 and are done using standard procedures (Sambrook, J. et al.,
  • the embodiment described utilizes the alpha portion of lacZ with lad.
  • the complete lacZ can also be used by providing a means to maintain complete repression by lad until induction is desired. This can be done in a variety of ways including control of AM15 laZ expression by a lambda specific promoter (PR') which prevents lacZ expression in the host __ coli until several minutes following infection by the bacteriophage, allowing lad levels to build up to suitable levels to enable complete repression. Additionally, low levels of lac repressor can be maintained in the host to assist in repression by lad until induction occurs, either by a mutation in lad or by addition of IPTG to the system.
  • a third alternative is to use an altered lad gene which gives rise to a repressor protein with higher specific activity, thereby allowing stronger repression of ⁇ - galactosidase production.
  • the source of starting materials for the cloning procedures are as follows: the pBluescript II SK+ and SK-, pBS(+), lambda gtll, and lambda L2B are available from Stratagene Cloning Systems, La Jolla, CA. Lambda L47.1 and pPreB: Short, J.M. , et al., Nucleic Acids Res.. 16:7583-7600. pMJR1560 is available from Amersham Corp., Arlington Heights, Illinois.
  • Lambda ZAP is a lambda phage vector which permits in vivo excision of inserts from the lambda vector to a plasmid. This is possible because the lambda phage contains the two halves of an fl bacteriophage origin of replication. In the presence of proteins supplied by fl helper phage, all DNA present between the two partial fl origins is automatically excised from the lambda phage.
  • the two halves come together to form an intact fl origin.
  • the resulting phagemid contains a Col El origin of replication and an ampicillin resistance gene, thus the insert is effectively subcloned into a plasmid vector. All sequences between the two partial fl origins are excised as a plasmid within hours.
  • these fl origins are positioned so that the lad gene can be automatically excised from the lambda phage from the mouse genomic DNA. Following this conversion from phage to plasmid, the insert may be rapidly sequenced or characterized by other known methods. Characterization of a large number of mutations within the la gene can be completed within 3 days following isolation of mouse genomic DNA, as opposed to several months using standard techniques.
  • a lambda ZAP is used to convert the test DNA inserts from integration in the lambda vector to a plasmid.
  • Other systems may also be used which allow excision and recircularization of a linear sequence of DNA thereby providing a rapid means with which the test DNA sequence may be transferred from the phage to a form suitable for analysis.
  • Such other systems include, but are not limited to, the use of FLP-mediated (Senecoff, J. et al., Proc. Natl. Acad. Sci. USA, 82:7270-7274 (1985); Jayaram, M. , Proc. Natl. Acad. Sci. USA. 82:5875-5879 (1985); McLeod, M.
  • test DNA sequences include (but are not limited to) : the lac I repressor, the cl repressor, any antibiotic resistance gene sequence (ampicillin, kanamycin, tetracycline, neomycin, chloramphenicol, etc.), the lambda red and gam gene sequences, a thymidine kinase gene sequence, a xanthine-guanine phosphoribosyl transferase gene sequence, sequences that code for restriction enzymes or methylation enzymes, a gene sequence that codes for luciferase, and/or a tRNA stop codon or frameshift suppressor gene sequence.
  • test sequence(s) By bracketing the test DNA sequence(s) with convenient restriction sites, as shown in Figure 2, the test sequence(s) can be separated away from the mouse DNA with restriction enzymes and subsequently ligated with lambda or cosmid vectors which contain cos sites or if the test sequence is linked to a replication origin it can be transformed directly. Background can be reduced in such a system by including with the test DNA sequences a sequence that is necessary for lambda phage replication, which is then cloned with the test DNA sequence into a lambda genome deficient or defective in that sequence.
  • Lambda LIZ alpha The modified Lambda genome, designated Lambda LIZ alpha, is prepared through a series of molecular gene manipulations as diagrammed in Figures 4 through 8.
  • Figure 4 depicts the construction of pBlue
  • pBluescript SK- (Stratagene, La Jolla, California) is modified using site directed mutagenesis to introduce an Ava III restriction site at a position 5' to the open reading frame for the Lad gene, but downstream from the ampicillin resistance gene and the ColEl origin of replication present on pBluescript to form pBlue MI-.
  • FIG. 5 depicts the construction of pLadq.
  • pBluescript II SK+ (Stratagene) is digested with the restriction enzymes PstI and EcoRI, both which cleave in the polylinker region to form linearized pBluescript SK+ lacking the small fragment derived from the polylinker.
  • pMJR1560 (Amersham Corporation, Arlington Heights, Illinois) is digested with the restriction enzymes PstI and EcoRI to release a Laclq- containing fragment that is separated by agarose gel electrophoresis and eluted from the gel. The laclq- containing fragment is then ligated into the linearized pBluescript SK+ to form pLadq.
  • Figure 6 depicts the construction of plnt.l.
  • a double stranded DNA segment defining multiple cloning sites (a polylinker) is produced by synthetic olignucleotide synthesis and annealing.
  • the polylinker contains multiple restriction endonuclease recognition sequences including two Ava III sites flanking Xbal, Kpnl and Pvul sites.
  • the polylinker is digested with Ava III to form Ava III cohesive termini on the polylinker.
  • pBlueMI- is digested with Ava III and the polylinker is ligated into pBlueMI-to introduce the Pvul site into the Ava III site and form plnt.l
  • FIG. 7 depicts the construction of pPreLadqZ (pPRIAZ) .
  • pPre B is first prepared as described by Short et al, Nucl. Acids Res.. 16:7583-7600 (1988).
  • plasmid pUC 19 ATCC
  • pJF3 was digested with Hindlll, dephosphorylated and ligated to complementary oligonucleotides having Hindlll-compatible ends and defining a T3 RNA polymerase promoter as described by Morris et al. Gene. 41:193-2000 (1986).
  • a resulting plasmid, designated pBluescribe (pBS) was isolated that contained the T3 promoter oriented to direct RNA synthesis towards the multiple cloning site of pUC 19.
  • pBS was then digested with Aatll and Narl, treated with mung bean nuclease and alkaline phosphatase, and ligated to a 456 base pair (bp) Rsal/Drall blunt-end fragment isolated from the pEMBL8 plasmid described by Dente et al, Nucl.Acids Res.. 11:1645-1655 (1983).
  • the 456 bp fragment contains the intergenic region of fl phage, but does not contain the fl gene II promoter sequence.
  • Phagemid clones were isolated from the resulting ligation mixture and clones were isolated containing both orientations (+ or -) of the intergenic region and are designated pBS(+) or pBS(-), where "+" indicates that the intergenic region is in the same orientation as the lacZ gene.
  • pBluescript SK(-) and SK(+) were produced from pBS(-) and pBS(+), respectively, by digestion of each with EcoRI and Hindlll, followed by blunt ending with Klenow fragment of DNA polymerase I. The blunt-ended molecules were ligated to a blunt-ended synthetic polylinker containing 21 unique restriction sites as described by Short et al, Nucl.Acids Res..
  • the synthetic oligonucleotide and the Rsal/Hinfl fragment were ligated with a 3009 bp Dral/Ndel fragment obtained from pBS to form the plasmid pBST-B.
  • the initiator domain of the fl intergenic region was separately cloned by digesting pEMBL8 with Sau961 and Dral to form a 217 bp fragment that was then blunt- ended with Klenow and then subcloned into the Narl site of pBST-B to form pBSIT0#12.
  • pBluescript SK(-) was digested with Nael and partially digested to cut only at the Pvul site located adjacent to the fl origin, and the resulting fragment lacking the fl origin was isolated. The isolated fragment was ligated to the Nael/Pvul fragment of pBSITO#12 that contains the terminator and initiator regions of the fl intergenic region to form plasmid pPre B.
  • Lambda gtll (ATCC #37194) was digested with Kpnl and Xbal to produce a 6.3 kilobase (kb) fragment containing the LacZ gene, which was agarose gel purified.
  • pBS(+) prepared above and available from Stratagene was digested with Kpnl and Xbal, and the resulting LacZ fragment was ligated into pBS(+) to form pBS(LacZ).
  • pLadq from above was digested with Narl and Sal I and the resulting small fragment containing the Laclq gene was isolated.
  • pBS(LacZ) was digested with Narl and Sail and the resulting large fragment containing the LacZ gene was isolated and ligated to the small Ladq-containing fragment to form pLadqZ.
  • plnt.l prepared above was digested with Kpnl, and the resulting linear molecule was ligated to the Lad-LacZ fragment, produced by digesting pLadqZ prepared above with Kpnl, to form plntLa qZ.
  • plntLadqZ was then digested with Xbal, blunt-ended with Klenow, digested with Seal, and the resulting large fragment containing LacZ-LacIq and most of the ampicillin resistance gene was isolated.
  • pPre B prepared above was digested with Pvul, blunt ended with mung bean nuclease, digested with Seal and the resulting fragment containing the terminator and initiator fl intergenic region components was isolated and ligated to the plfntLadqZ-derived large fragment to form plasmid pPreLadqZ (pPRIAZ)
  • Figure 8 shown in two panels 8A and 8B, depicts the construction of Lambda LIZ alpha.
  • Lambda L47.1 described by Loenen et al, Gene. 10:249-259 (1980), by Maniatis et al, in "Molecular Cloning: a Laboratory Manual” , at page 41, Cold Spring Harbor, New York (1982), and by Short et al, supra. and having the genetic markers (srllambdal- 2)delta, imm434 cl-, NIN5, and chi A131, was digested with EcoRI, Hindlll and Smal to form a Lambda L47.1 digestion mixture.
  • Lambda L2B available from Stratagene, was first digested with Xbal, then treated with Klenow to fill-in the 5' Xbal overhang, then digested with Mlul to form a L2B digestion mixture.
  • the L47.1 and L2b digestion mixtures were ethanol- precipitated to prepare the DNA for ligation, and were then ligated to pPRIAZ prepared above that had been linearized with Ndel to form Lambda LIZ alpha.
  • Lambda LIZ alpha is a preferred modified Lambda bacteriophage for use in the present invention because it combines the elements of 1) a reporter gene in the form of the alpha component of LacZ, 2) the lambda bacteriophage excision capability provided by the presence of the cos sites at the termini, 3) an indicator gene system in the form of the Laclq target gene, including a lad promoter and the repressor structural gene sequences, and 4) an fl origin of replication arranged according to the in vivo excision system of Short et al, supra. that allows quick isolation of the mutated test gene from positive colonies containing the mutated test gene for sequencing.
  • PBLs Peripheral blood lymphocytes
  • ml venous blood drawn from volunteer donors.
  • PBS phosphate buffered saline
  • Lymphocytes are also isolated from tonsils obtained from therapeutic tonsillectomies from consenting patients. The tonsils are first homogenized and then lymphocytes are isolated over Histopaque as described above.
  • SCID mice having the autosomal recessive mouse mutations scid are obtained from Imdyme (San Diego, California) .
  • SCID mice are derived from an inbred strain of mice, C.B-17 (Balb/c- C57BL/Ka-Igh-lb/ICR (N17F34) as described by Bosma et al.. Nature, 301: 527-530 (1983).
  • mice lacking IgM, IgGl or IgG2a determined that the defect was inheritable and under the control of the recessive scid gene. Bosma et al., supra. A colony of mice can be established which are homozygous for the defective gene.
  • the SCID mice are maintained in microisolator cages (Lab Products, Maywood, New Jersey) containing sterilized food and water.
  • C. Preparation of SCID/hu Chimeras SCID mice obtained in Example 6b are reconstituted by intraperitoneal injection with at least 5 X 10 7 human PBLs or tonsil lymphocytes prepared in Example 6a.
  • the recipient SCID mice are designated SCID/hu chimeras which contain the subject rDNA.
  • the human PBL reconstituted SCID mouse model is then used for assaying the effects of mutagens on human cells as described in this invention.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Analytical Chemistry (AREA)
  • Hematology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Veterinary Medicine (AREA)
  • Environmental Sciences (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Animal Husbandry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Plant Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Analyse permettant de contrôler et d'estimer le potentiel mutagène d'agents consistant à créer des animaux transgéniques porteurs d'une ou de plusieurs séquences d'ADN test pouvant être rapidement récupérées et examinées afin de déterminer des mutations, après exposition à un ou à plusieurs agents soupçonnés d'être mutagènes.
EP91908707A 1990-04-05 1991-04-05 Test de mutagenese a l'aide d'animaux transgeniques porteurs de sequences d'adn tests Withdrawn EP0608210A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US50567690A 1990-04-05 1990-04-05
US505676 1990-04-05
PCT/US1991/002364 WO1991015579A1 (fr) 1990-04-05 1991-04-05 Test de mutagenese a l'aide d'animaux transgeniques porteurs de sequences d'adn tests

Publications (2)

Publication Number Publication Date
EP0608210A4 EP0608210A4 (fr) 1993-06-07
EP0608210A1 true EP0608210A1 (fr) 1994-08-03

Family

ID=24011339

Family Applications (1)

Application Number Title Priority Date Filing Date
EP91908707A Withdrawn EP0608210A1 (fr) 1990-04-05 1991-04-05 Test de mutagenese a l'aide d'animaux transgeniques porteurs de sequences d'adn tests

Country Status (4)

Country Link
EP (1) EP0608210A1 (fr)
JP (1) JPH06506103A (fr)
CA (1) CA2079792A1 (fr)
WO (1) WO1991015579A1 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9123929D0 (en) * 1991-11-11 1992-01-02 Wellcome Found Novel expression system
EP0671955A4 (fr) * 1992-02-14 1997-04-09 Stratagene Inc Test de mutagenese utilisant des animaux non humains transgeniques portant des sequences d'adn test.
DE4228162C1 (de) * 1992-08-25 1994-01-13 Rajewsky Klaus Dr Verfahren zum Ersetzen homologer Genabschnitte aus Säugern in der Keimbahn von nicht-menschlichen Säugern
DE69926629T2 (de) 1998-05-31 2006-06-08 University Of Georgia Research Foundation, Inc. Transgene fische, die ein von bakteriophagen abgeleitetes transgenes konstrukt zum nachweis von mutationen tragen
AU1323000A (en) 1998-10-26 2000-05-15 University Of Georgia Research Foundation, Inc., The Transgenic fish carrying plasmid for mutation detection and methods

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0289121A2 (fr) * 1987-05-01 1988-11-02 Stratagene Test de mutagénèse utilisant des êtres non humains transgéniques porteurs des séquences d'ADN à essayer
EP0370813A2 (fr) * 1988-11-25 1990-05-30 Exemplar Corporation Dosage rapide par criblage de mutagénèse et tératogénèse
WO1992017605A1 (fr) * 1991-04-02 1992-10-15 Ingeny B.V. Procede de detection de mutations, mammifere transgenique, cellule transgenique de mammifere, et procede de recherche de proprietes mutagenes dans des agents ou des milieux

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4736866B1 (en) * 1984-06-22 1988-04-12 Transgenic non-human mammals

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0289121A2 (fr) * 1987-05-01 1988-11-02 Stratagene Test de mutagénèse utilisant des êtres non humains transgéniques porteurs des séquences d'ADN à essayer
EP0370813A2 (fr) * 1988-11-25 1990-05-30 Exemplar Corporation Dosage rapide par criblage de mutagénèse et tératogénèse
WO1992017605A1 (fr) * 1991-04-02 1992-10-15 Ingeny B.V. Procede de detection de mutations, mammifere transgenique, cellule transgenique de mammifere, et procede de recherche de proprietes mutagenes dans des agents ou des milieux

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF CELLULAR BIOCHEMISTRY. vol. SUPPL, no. 13B, 21 January 1989, NEW YORK, USA page 184 SHORT, J.M. ET AL. 'The effects of eukaryotic methylation on recovery of a Lambda phage shuttle vector from transgenic mice' *
NUCLEIC ACIDS RESEARCH. vol. 18, no. 10, 25 May 1990, ARLINGTON, VIRGINIA US pages 3007 - 3013 KOHLER, S.W. ET AL. 'Development of a short-term, in vivo mutagenesis assay: the effects of methylation on the recovery of a lambda shuttle vector from transgenic mice' *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 88, no. 18, September 1991, WASHINGTON US pages 7958 - 7962 KOHLER, S.W. ET AL. 'Spectra of spontaneous and mutagen-induced mutations in the lacI gene in transgenic mice' *
See also references of WO9115579A1 *

Also Published As

Publication number Publication date
EP0608210A4 (fr) 1993-06-07
WO1991015579A1 (fr) 1991-10-17
JPH06506103A (ja) 1994-07-14
CA2079792A1 (fr) 1991-10-06

Similar Documents

Publication Publication Date Title
US5510099A (en) Mutagenesis testing using transgenic non-human animals carrying test DNA sequences
US11849709B2 (en) Genetically modified rat models for severe combined immunodeficiency (SCID)
US5347075A (en) Mutagenesis testing using transgenic non-human animals carrying test DNA sequences
Wilmut et al. Genetic manipulation of mammals and its application in reproductive biology
US20070204354A1 (en) Integrated in vivo animal experimentation systems
EP0565638A1 (fr) Animaux non humains susceptibles d'avoir une tumeur
US5919997A (en) Transgenic mice having modified cell-cycle regulation
Torres 5 The Use of Embryonic Stem Cells for the Genetic Manipulation of the Mouse
EP0671955A1 (fr) Test de mutagenese utilisant des animaux non humains transgeniques portant des sequences d'adn test
WO1991015579A1 (fr) Test de mutagenese a l'aide d'animaux transgeniques porteurs de sequences d'adn tests
US6221647B1 (en) Efficient construction of gene targeting using phage-plasmid recombination
US5824287A (en) Mutagenesis testing using transgenic non-human animals carrying test DNA sequences
Chen et al. Inducible gene trapping with drug-selectable markers and Cre/loxP to identify developmentally regulated genes
Skynner et al. Transgenic mice ubiquitously expressing Human Placental Alkaline Phosphatase (PLAP): an additional reporter gene for use in tandem with�-galactosidase (lacZ)
US6472583B1 (en) Plasmid-based mutation detection system in transgenic fish
WO1989009272A1 (fr) Souris transgenique pour la mesure et la caracterisation de mutations induites in vivo
WO1993017123A1 (fr) Test de la mutagenicite a l'aide de genes rapporteurs avec frequences de methylation modifiees
CA2264450A1 (fr) Methodes de surveillance de chromosomes sexuels heterologues
Valancius-Mangel et al. POTENTIAL USES OF TRANSGENIC AND GENE-TARGETED ANIMALS IN TOXICOLOGIC RESEARCH
JPH1056915A (ja) Ｌｄｌレセプター遺伝子不活性化動物
Genes Inducible Gene Trapping with
BOTQUIN¹ et al. AND ANGEL CID-ARREGUI 3

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19921104

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LI LU NL SE

17Q First examination report despatched

Effective date: 19980825

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20001116