EP0671955A4 - Test de mutagenese utilisant des animaux non humains transgeniques portant des sequences d'adn test. - Google Patents

Test de mutagenese utilisant des animaux non humains transgeniques portant des sequences d'adn test.

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Publication number
EP0671955A4
EP0671955A4 EP93906965A EP93906965A EP0671955A4 EP 0671955 A4 EP0671955 A4 EP 0671955A4 EP 93906965 A EP93906965 A EP 93906965A EP 93906965 A EP93906965 A EP 93906965A EP 0671955 A4 EP0671955 A4 EP 0671955A4
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EP
European Patent Office
Prior art keywords
lambda
dna sequence
test dna
gene
terminator
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Withdrawn
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EP93906965A
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German (de)
English (en)
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EP0671955A1 (fr
Inventor
Jay M Short
Patricia L Kretz
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Stratagene California
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Stratagene California
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Publication of EP0671955A1 publication Critical patent/EP0671955A1/fr
Publication of EP0671955A4 publication Critical patent/EP0671955A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • 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
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C12N15/72—Expression systems using regulatory sequences derived from the lac-operon
    • 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.
  • DNA alterations that are caused by potential mutagenic agents have generally been approached by performing studies on procaryotic or eukaryotic cells in culture (in vitro tests).
  • the well-known Ames' test uses a special strain of bacteria to detect these mutations. Ames et al, 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.
  • 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 re-isolated, propagated in bacteria, and analyzed for mutations. Because the host is only a mammalian cell line and not a live animal, 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.
  • 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 organism and methods utilizing such organisms for mutagenesis testing, satisfies these needs. More specifically, the present invention provides a sensitive screen for the utagenicity 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 organisms which must be used for mutagenesis testing.
  • the present invention contemplates a method for assaying the mutagenic potential of an agent.
  • the method generally comprises administering a predetermined amount of the agent to an organism containing cells having a genome characterized by the presence of a target gene system containing a test DNA gene sequence that is capable of detection by bioassay in a host cell upon mutation of the test DNA sequence. After a predetermined exposure time period, a predetermined amount of the target DNA system is then recovered from cells harvested from the exposed organism. The recovered target gene system is then introduced into and expressed in a restriction system deficient host cell, whereupon mutation of the target gene system is determined in bioassay.
  • the invention describes a mutagenesis testing method comprising: (a) exposing a transgenic non-human organism to a test agent, wherein the transgenic organism comprises somatic and germ cells containing a test DNA sequence that is capable of detection in bioassay by expression of a test DNA sequence gene product; and
  • the host cell contains a reporter gene transcription unit controlled by a promoter that is regulated by the test DNA sequence gene product.
  • the reporter gene product confers a selective growth advantage to the bioassay system, thereby allowing selectable detection of the mutated test DNA sequence over non-mutated test DNA sequences in the target gene system.
  • the organism is a transgenic plant or animal, such as a transgenic fish or rodent, and preferably is a transgenic rat or mouse.
  • Preferred methods have been developed which use a target gene system comprised of a set of two genes, a test gene and a reporter gene.
  • the test gene is incorporated into an organism or its somatic and germ cells to screen for compounds having mutagenic, carcinogenic or teratogenic activity.
  • the reporter gene can be present in a separate assay system such as a prokaryotic or eukaryotic host cell, or it can be incorporated in the organism with the test gene. Exposure of the organism or its cells to compounds having any of these activities causes mutations resulting in alterations in expression of the test gene. Mutations in the test gene are measured by detecting reporter gene expression, which is affected by test gene expressions.
  • Both the test gene and the reporter gene constructs are each operatively linked to a promoter, preferably a prokaryotic promoter.
  • transgenic non-human organisms containing a target gene system of this invention for practicing the mutagenesis testing methods described herein.
  • host cells that contain a reporter gene transcription unit for detecting a mutated test DNA sequence in a bioassay of this invention.
  • 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.
  • Other permutations of this improved method are described further herein.
  • FIG. 1 illustrates the sequence of process steps for performing the invention.
  • Figure 2 illustrates an alternative method for recovering the transgenic test DNA sequence.
  • Figure 3 illustrates in two panels (3A and 3B) a
  • Figure 4 illustrates a schematic depicting the construction of plasmid pBlue MI-.
  • Figure 5 illustrates a schematic depicting the construction of plasmid pLacI q-
  • Figure 6 illustrates a schematic depicting the construction of plasmid plnt.l.
  • Figure 7 illustrates a schematic depicting the construction of plasmid pPreLacIqZ.
  • Figure 8 illustrates in two panels ( Figure 8A and Figure 8B) a schematic depicting the construction of phage Lambda LIZ Alpha.
  • Figure 9 is a graph that illustrates the mutation frequency for spontaneous (closed symbols) and induced (open symbols) mutations as described in Example 8.
  • Figure 10 is a schematic in two panels ( Figure 10A and Figure 10B) illustrating a gene activating selectable system as described in Example 9.
  • Figure 10A shows the operation of the system under the condition where a wild-type (non-mutated) lad gene present on the Lambda LIZ Alpha vector (upper vector) represses the reporter gene transcription unit (lower vector) , thereby inactivating groE gene expression and inhibiting plaque formation.
  • Figure 10B shows the operation of the system under the condition where a mutated lad gene present on the Lambda LIZ Alpha vector (upper vector) does not represses the reporter gene transcription unit (lower vector) , thereby activating groE gene expression and allowing plaques to form.
  • Figure 11 is a schematic in two panels ( Figure 11A and Figure 11B) illustrating a gene inactivating selectable system as described in Example 10.
  • Figure 11A shows the operation of the system under the condition where a wild-type (non-mutated) lacl gene present on the Lambda LIZ Alpha vector (upper vector) represses the reporter gene transcription unit (lower vector) , thereby activating S 5 gene expression and inhibiting plaque formation.
  • Figure 11B shows the operation of the system under the condition where a mutated lacl gene present on the Lambda LIZ Alpha vector (upper vector) represses the reporter gene transcription unit (lower vector) , thereby inactivating S 5 gene expression and allowing plaques to form.
  • the present invention contemplates engineered somatic and germ cells of an organism, such as an animal, animal embryos or differentiated animals, having a genome characterized by the presence of a target gene system useful for the testing of mutagenic potential of a suspected mutagen.
  • the target gene system is recoverable from the test organism's genome by nucleotide sequences that define an excision means flanking the target gene system, such as an excisably integrated genetic element.
  • 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 for expression of a test gene product.
  • the recoverable target gene system is included within excisably integrated lambda phage DNA.
  • the target gene system need not be included within lambda phage DNA to be recoverable for purposes of the present invention.
  • the target gene system can be present within a plasmid, cosmid, filamentous phage, or present in the genome of the animal and be recoverable.
  • test gene refers to a sequence of nucleotides which are the direct target for mutation by a suspected mutagen.
  • reporter gene refers to a nucleotide sequence which expresses a detectable phenotype in an assay system, and the expression of which reporter gene is controlled by the test gene.
  • 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.
  • a recombinant DNA molecule (rDNA) of this invention (subject rDNA) is used for preparing a non- human transgenic organism of this invention and contains a target gene system as described herein.
  • a target gene system preferably is operatively linked to a lambda phage and can be comprised of any of a variety of test genes (test DNA sequence) whose transcription results ultimately in a detectable phenotype or genotype where a mutation in the nucleotide sequence of a test gene measurably alters the detectable phenotype or genotype.
  • 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 test gene can be selected from the group of nucleotide sequences which encode regulatory molecules that bind to a sequence controlling reporter gene expression. These can be repressers or other regulatory molecules, including anti-sense RNA.
  • a preferred test gene is a repressor or activator gene whose expression product directly alters the detectable expression of a reporter gene.
  • Exemplary is the repressor protein encoded by the lacl gene, and genetic variants of the lacl gene that function to block transcription of the beta-galactosidase gene (lacZ) by binding to the operator region of the lacZ gene's expression signals.
  • lacZ is a reporter gene.
  • a preferred lacl test gene is the laclq variant that expresses eight- to ten-fold elevated levels of repressor protein and more tightly represses expression of the lacZ reporter gene when under the control of a lac operator.
  • the inactivation of the lac repressor gene by a mutagenic event causes the transcription and translation of a defective repressor protein that is no longer able to repress expression of the lacZ reporter 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 test gene of the target gene system is operatively linked to a reporter gene, i.e., both test and reporter genes are linked on the same DNA molecule.
  • the reporter gene is present in a host cell of the assay system and is regulated by the expression of the test gene.
  • the reporter gene transcription unit is present in the host cell for bioassay, such as the E_;_ coli into which the recovered test DNA sequence is to be introduced, the test DNA sequence gene product acts in trans to influence the expression of the reporter gene product.
  • a reporter gene provides a means for detecting mutations in the test gene.
  • a reporter gene is a gene that encodes a detectable phenotype or genotype and whose expression is under the control of the test gene. Typically, the reporter gene is the final (endpoint) gene in a biochemical pathway initiated or regulated by the test DNA gene product.
  • the selection of reporter genes is based on the following criteria: (i) 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.
  • a reporter is lethal in the assay system, and in other systems the reporter is not lethal. Examples of a non-lethal reporter genes are genes which confer the ability for growth, amplification or replication of the reporter gene, or cells harboring the reporter gene.
  • any of a variety of genes can function as the reporter gene according to the present invention so long as the expressed reporter gene product is detectable.
  • Genes that encode detectable phenotypes include drug resistance markers, enzymes whose activity produces a detectable reaction product and the like.
  • Candidate enzymes include beta- galactosidase (Norton et al, Mol. Cell. Biol, 5:281- 290 (1985), peroxidase and luciferase (de Wet et al, Mol. Cell. Biol, 7:725-737 (1987) .
  • a preferred reporter gene is the E. coli 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 lacZAM15 gene product.
  • the phenotype produced by the reporter gene can result in detection based on a phenotypic selection such as a colori etric selection, growth selection, enzymatic activity, and the like.
  • Reporter genes which encode enzymes, antigens or other biologically active proteins which can be monitored easily by biochemical techniques are preferred.
  • a reporter gene is expressed when the test gene is not mutated, and is not expressed when the test gene is expressed as a functional protein.
  • a reporter gene can be expressed only when the test gene is mutated.
  • the reporter gene confers a growth advantage to the assay system containing the mutated test gene, and the reporter gene thereby provides a mechanism to select for a detection of the mutation event.
  • a growth advantage in this context, is provided to the reporter gene, whether it replicates (grows) and is amplified in a host cell or in the form of an autonomous genetic element, such as the exemplary phage.
  • the occurrence of a mutation in the test gene is selected for, thereby increasing the efficiency of the system to detect mutagenic activity.
  • Such a system is referred to herein as a "selectable system”.
  • a reporter gene is under the transcriptional control of an operator that is repressed by a test gene product, i.e. the test gene product is a repressor.
  • the test gene product is a repressor.
  • the test gene loses its repressor function, and the reporter transcription unit is expressed providing a growth advantage to the assay system.
  • a preferred system uses a lac operator controlling reporter gene transcription, and uses a lac repressor such as lacl or lacl q as the test gene.
  • Preferred reporter genes that can be used to confer a selective growth advantage include groE and lambda S 5 as described herein, beta-galactosidase- based genes such as lacZ or alpha-lacZ, and E. coli gene that are essential for lambda DNA replication, but dispensable for E. coli, such as grpD, grpE or cro. See, for example, "Lambda II" Hendrix et al, eds., Cold Spring Harbor Press, 1983, p.147. Additional selectable genes which confer a useful growth advantage as reporter genes are amino acid genes, and tRNA genes
  • the groE gene is an E. coli gene that is required for lambda phage particle morphogenesis.
  • the groE operon is actually two closely linked genes that encode the GroEL and GroES proteins required for lambda phage head assembly. Mutations in groEL or groES genes block lambda head assembly at an early stage.
  • a mutant E. coli host is utilized for reading the mutagenesis assay that has a deficiency in groEL, groES, or both, where defects in both is designated groESL.
  • the wild type groE, the groEL or groES gene, or both (groESL, is/are supplied by the reporter gene's transcription unit, and upon expression confers the ability (selective advantage) to assemble phage particles.
  • Preferred GroE systems are described in Examples 9 and 11.
  • the expression of a groE reporter gene product is activated by the introduction of a mutated lacl test gene product into a host reporter system, which allows bacteriophage plaques to form, thereby indicating the presence of the mutated test gene in the host.
  • a host reporter system which allows bacteriophage plaques to form, thereby indicating the presence of the mutated test gene in the host.
  • Such a system is referred to as an "activating reporter gene system" because the activation of the reporter gene produces the detectable event.
  • S 5 a mutated lambda S gene product designated S 5 .
  • the S 5 gene product prevents plaque formation by inhibiting the formation of a functional inner membrane pore through which phage particles can extrude during morphogenesis.
  • the efficient expression of the S 5 phenotype requires that the reporter gene be expressed in E. coli which is supF.
  • the gene is referred to as a dominant negative inactivating gene because its effect is dominant, not recessive, and because its expression is a negative marker, i.e., it inhibits plaque formation.
  • the amino acid residue sequence of a lambda S gene is coded for in the wild-type lambda genome at nucleotide base residues 45186 to 45006.
  • the complete nucleotide sequence of wild-type lambda is well known, and is also described in "Lambda II" by Hendrix et al., Cold Spring Harbor Press, 1983.
  • the S 5 mutations have been identified to be the substitution of an adenine (A) for a thymidine (T) at nucleotide 45214, and the substitution of an adenine (A) for a cytosine (C) at nucleotide 45310.
  • S 5 mutations can thus readily be prepared by synthetic methods such as oligonucleotide synthesis and hybridization of the synthesized oligonucleotides to form a complete gene, as is well known in the art.
  • a preferred S 5 gene for use in the invention has the nucleotide sequence shown in SEQ ID NO 18.
  • a preferred S 5 system is described in Example 10.
  • the expression of a S 5 reporter gene product is inactivated by the introduction of a mutated lacl test gene product, which allows bacteriophage plaques to form, thereby indicating the presence of the mutated test gene.
  • a mutated lacl test gene product which allows bacteriophage plaques to form, thereby indicating the presence of the mutated test gene.
  • Such a system is referred to as an "inactivating reporter gene system” because the inactivation of the reporter gene produces the detectable event.
  • the inactivating reporter gene system utilizes competing transcripts as described further in Example 10 as exemplary. Any other dominant negative inactivating gene can be utilized in a reporter gene transcription unit according to the system described herein for the S 5 gene.
  • the test gene is operatively linked to expression signals to facilitate the rapid detection of mutations by the present invention. The type of expression signals depends upon the host cell in which the reporter gene is bioassayed.
  • a preferred host is a prokaryotic cell, and therefore the reporter is preferably under the control of prokaryotic expression signals.
  • the test gene system Upon recovery of the target gene system, e.g., by one of the various excision means described herein, 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 lacl, lacl q , lacl sq or lacl c ⁇ gene and includes a lacl promoter region.
  • the bacterial lac operator-repressor system is preferred because it is one of the most basic and thoroughly studied examples of a protein-nucleic acid interaction that regulates transcription of a gene, as described by Coulondre et al, Mol. Biol. , 117:577 (1977) , Miller, Ann. Rev. Genet. , 17:215 (1983) ; and in "The Operon", Miller et al, eds., Cold Spring Harbor, 1980.
  • 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 et al, Cell, 48:555 (1987), and Brown et al, Cell, 49:603 (1987).
  • IPTG isopropyl beta-D thiogalactoside
  • An important difference between previous uses of the lac operator-repressor system and the present method is that mutation rather than induction is used to derepress the reporter genes to express protein whose function is solely to serve as an indicator.
  • the target gene system is excisable as an infectious lambda phage.
  • 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.
  • 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) .
  • Excision elements can be site-specific restriction endonuclease nucleotide sequences, or can be other genetic elements that facilitate site-specific excision.
  • Preferred excision element nucleotide sequences are lambda cos sites, flp recombinase recognition sites, loxP sites recognized by the Cre protein, and the first and second halves of the filamentous bacteriophage (M13, ff or fl) origin of replication (referred to generically as an fl bacteriophage origin of replication) , 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 cos site nucleotide sequences when utilizing lambda bacteriophage in vitro packaging extracts as described herein.
  • it is considered useful to boost the packaging reaction by the repeated addition of aliquots of packaging extract to the packaging reaction, as the extract becomes depleted during the packaging reaction, which can be remedied by multiple additions of extract.
  • the excision elements of a target gene system confer the ability to readily recover the target gene system from the mutagen 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 lacl q 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, lacl 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.
  • mRNA messenger ribonucleic acid
  • the operator sequence has to be built into the reporter gene at the location between the transcription initiation site and the initiation codon ATG.
  • 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 "8 molar (M) , preferably greater than 10 "9 M, and more preferably greater than 10 "10 M.
  • M molar
  • 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.
  • lac repressor The operator for the lac repressor has been well characterized and is used as exemplary herein. See Miller et al, in "The Operon", Cold Spring Harbor Laboratory, New York (1980) , for a detailed study. Alternative nucleotide sequences have been described for a lac repressor operator that specifically binds to repressor. See, for example, the description of numerous lac operator variants and the methods for characterizing their repressor-binding activity reported by Sartorius et al, EMBO J.. 8:1265-1270 (1989) ; and Sadler et al, Proc. Natl. Acad. Sci. USA, 80:6785-6789 (1983) .
  • nucleotide sequence that binds lac repressor specifically can be used in the present invention, although wild type and optimized "ideal" operators are preferred and used as exemplary herein.
  • the original lac operator sequence (5'- GGAATTGTGAGCGGATAACAATCC-3 ⁇ ; SEQ ID NO 1), or a mutant lac operator which binds repressor eight times tighter and has the sequence (5 '-ATTGTGAGCGCTCACAAT-3 • ; SEQ ID NO 2) , are preferred for use in vector construction.
  • Two preferred optimized operators derived from the lac operon include the nucleotide sequences as follows:
  • 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.
  • 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.
  • Typical of such vector plasmids 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 detectable end point for the bioassay can be either lytic plaque formation or a lysogenic phenotype, depending on the manner in which the genetics of the test DNA sequence and reporter gene are designed.
  • the resulting lysogen or lytic plaque can be designed to exhibit a color screen as the detection means, as shown herein.
  • the combined phenotype for the reporter gene of a growth advantage and color indication is contemplated.
  • a representative combined phenotype reporter gene system is one where the test gene controls two separate reporter genes; one providing a colorimetric phenotype, such as lacZ f and one providing the growth advantage, such as an activating reporter gene system.
  • An exemplary combined phenotype system is the groE system described in Example 9. The system provides a particular advantage in circumstances where there is a leaky gene. For example, the system can exhibit a low level of false positives in the form of a growth advantage to a reporter gene, but that false positive is deter inable as a false positive based on the color phenotype.
  • the reporter gene in the host cell can be present in a variety of forms.
  • the reporter gene can be present as a host cell genomic element, or as a transcription unit on a phage genome within the cell or on a plasmid, such as an F 1 plasmid, within the cell.
  • Still further embodiments contemplate methods for providing further regulation of the expression of the reporter gene, including methods for preventing expression of the reporter gene transcription unit until the test gene is introduced to methods involving the tight regulation of the reporter gene transcription unit.
  • the invention contemplates the use of
  • reporter structural gene transcription units in which the reporter structural gene is reversed within the reporter gene transcription unit relative to its promoter such that it cannot be expressed in the reverse order.
  • the reporter structural gene flips around to position in the correct orientation and therefore is under expression control of the reporter gene transcription unit's promoter.
  • Reversibility of the structural gene can be accomplished in a number of ways.
  • the structural gene can be flanked by nucleotide sequences defining flp sites which are activated by flp recombinase.
  • the reporter structural gene flips over and can be transcribed by the transcription unit.
  • the structural gene can be flanked by nucleotides defining the Cre-lox system, and can be flipped upon introduction of means for initiating Cre- lox mediated recombination. Still further, one can flank the structural gene by the att nucleotide sequence of lambda integrase, and flip the structural gene by introduction of lambda integrase.
  • Another mehcanism for closer regulation of the reporter gene transcription unit is to include a second repressor binding site to the transcription unit which is regulated independently from the test gene product.
  • the second repressor is inducible, and derepression of the second repressor is controlled by the addition of an inducer of the second repressor which is selected to act independently of the test DNA gene product.
  • An additional mechanism for regulation of reporter gene transcription is to include transcription or translation modifiers in the reporter gene transcription unit which repress translation until the test DNA gne is introduced into the host cell.
  • transcription or translation modifiers include the addition of a poly A transcription terminator after the reporter structural gene to stabiliize the reporter gene transcripts.
  • transcriptional modifiers include the introduction of various ribosome binding sites known to effect the efficiency of transcription, or the alteration of the nucleotide sequence around the ATG start site for translation to alter the effeciency of translation.
  • host cell strains in which the endogenous levels of protease are inhibited would be useful for boosting the level of expression of the reporter gene product.
  • An E. coli host cell having a mutation in the Ion gene or hfl gene are preferred examples of mutations that would desirably reduce the amount of endogenous protease in the host cell.
  • the invention contemplates multi- level transcription control such as to regulat the reporter gene by a second transcription unit which, in turn, is regulated by the test DNA gene product.
  • the test DNA gene product expresses lad
  • the host cell contains a first transcription unit having the lac operator to which the lac repressor binds.
  • This first transcription unit expresses, for example, the T7 polymerase gene product, which, upon expression, binds to the T7 polymerase operator that is controlling expression of the reporter gene transcription unit.
  • Other permutations are readily apparent.
  • the invention contemplates the DNA molecules, plasmids, nucleotide sequences and the like that are utilized in the test DNA sequences and in the reporter gene transcription units described herein. Also contemplated are host cells containing the DNA molecules of this invention.
  • 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 organism thereafter bred to produce test systems.
  • substantially all of the cells will contain the test DNA sequence.
  • the test transgenic organism 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 organism.
  • the test DNA sequence may be transferred into a host cell containing a reporter gene transcription unit, although such recovery and transfer is not requisite, and assayed for mutations, allowing rapid examination of multiple tissue specific genetic mutations.
  • any organism suitable for mutagenic testing may be used as the starting organism.
  • the organism can be plant or animal, and a preferred and exemplary embodiment is a non-human mammal, preferably a rodent.
  • a preferred and exemplary embodiment is a non-human mammal, preferably a rodent.
  • 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, 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. Typically, 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, Mol. Cell. Biol.. 7:1276-1279 (1987).
  • the cellular level 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.
  • the copy number of the test DNA sequence in the genome of a transgenic mammal can be varied to increase the number of targets for the suspected mutagen.
  • the copy number of the reporter gene in the bioassay host cell can be varied to optimize the expression of the reporter gene product.
  • the promoter strength can be varied to optimize the expression of the reporter gene product.
  • 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.
  • 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 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 (recovered) 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 target DNA sequence is inserted into the genome or is present on an extra chromosomal element, 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 host cells, such as 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. The test DNA sequence is thereafter replicated on indicator plates or in selective media. In one embodiment, the test DNA sequence is grown in a host cell such as E. coli. A phenotype indicating mutation of the test DNA sequence will identify a mutated test DNA sequence. The ratio of mutated test DNA sequences to the total number of test DNA sequences is a measure of the mutagenicity of the agent and metabolites thereof.
  • 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 1,000 to about 10 6 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, recovered from the total genomic DNA of the test organism, e.g. , by using a lambda packaging extract deficient in restriction systems which recognize and deactivate foreign DNA.
  • the recovered test DNA sequences may then be transferred into and expressed by restriction system deficient host cells, having deficiencies as described further herein.
  • 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, such as a system that is contained by a bacteriophage genome that is readily rescued. 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 organism 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 gamete cells.
  • Preferred organisms include non-human mammals, birds, fish, gymnosperms and angiosperms.
  • the present invention contemplates a transgenic fish for the in vivo screening for mutagenic compounds.
  • Fish represent a category of animals of great interest for agricultural and ecological reasons in the context of water-borne mutagenic compounds, and provide a convenient system for screening mutagenic compounds in a variety of fish species including, but not limited to, trout, salmon, carp, shark, ray, flounder, sole, tilapia, medaka, goldfish, guppy, molly, platyfish, swordtail, zebrafish, loach, catfish, and the like.
  • Transgenic fish of numerous species have been prepared, providing the skilled practitioner with a variety of procedures for developing a transgenic fish having an excisably-integrated target gene according to the present invention. See, for example, the teachings of Ozato et al, Cell Differ.. 19:237-244 (1986), Inoue et al, Cell Differ. Dev.. 29:123-128
  • the present invention contemplates a non- human animal containing a modified lambda bacteriophage (rDNA) of the present invention excisably-integrated in the genome of the animal's somatic and germ cells, i.e., a transgenic animal.
  • rDNA modified lambda bacteriophage
  • transgenic mammals particularly preferred are transgenic mammals, and are utilized as exemplary herein.
  • a particularly preferred transgenic mammal is the transgenic mouse described herein that contains a single copy of the lambda LIZ Alpha vector system.
  • An embryo of the preferred transgenic mouse line containing the excisably integrated lambda LIZ Alpha phage vector was deposited with the American Type Culture Collection (ATCC) on March 17, 1992, under the ATCC accession number 72011.
  • Mammals containing a rDNA of the present invention are typically prepared using the standard transgenic technology described in Hogan et al, Manipulating the Mouse Embryo: 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. Production of transgenic mammals is 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.
  • 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 ..? 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. These 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.
  • rDNA 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, cyclost ⁇ mes, arthropods, insects, mollusks, thallaphytes, embryophytes including gymnosper s and angiosper s.
  • 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, Gasser et al, Science, 244:1293-1299 (1989); European Patent Application No. 0257472 filed August 13, 1987 by De La Pena et al; PCT Pub. No. WO 88/02405 filed October 1, 1987 by Trulson et al; PCT Pub. No. WO
  • 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.
  • 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
  • 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, Wagner et al, U.S. Patent No. 4,873,191 (Oct. 10, 1989) ; Hogan et al, Manipulating the Mouse Embryo: A Laboratory Manual, Cold Springs Harbor, New York (1987); Capecchi, Science, 244:288-292 (1989); and Luskin et al, Neuron 1:635-647 (1988).
  • 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 ste 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 ste 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 Embryo: A Laboratory Manual, Hogan et al, eds., Cold Spring Harbor, New York, (1986) .
  • the infection of cells within an animal using a replication incompetent retroviral vector has been described by Luskin et al, Neuron, 1:635-647 (1988).
  • 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.
  • the invention contemplates the use of mutagenesis testing systems according to the present invention which provide selection for mutagenic events.
  • Selectable systems are particularly preferred because (1) they provide increased speed and convenience (i.e., efficiency) in detecting the reporter gene product due to the selection of mutagenized test DNA sequences over a background of non-mutagenized test DNA sequences and may increase sensitivity of the assay, and (2) they allow for the screening of larger numbers of clones per unit of culture medium that is used to bioassay the mutagenic event.
  • test DNA sequences and reporter genes are contemplated by the present invention in which the reporter gene provides a selective growth advantage to the host cell or bacteriophage which harbors the mutagenized test DNA sequence.
  • the expression of the reporter gene in response to the test DNA gene product provides the switch between a selective growth advantage and a growth disadvantage.
  • the non-mutated test DNA gene product functions, directly or indirectly, to repress expression of the reporter gene product.
  • the non-mutated test DNA gene product functions, directly or indirectly, to de-repress (or activate/promote) expression of the reporter gene product.
  • the reporter gene transcription unit is independent of the test gene, and typically located within the host cell used for bioassaying the mutagenic event.
  • the regulation of the reporter gene by the test gene occurs between separate genetic elements and is referred to as regulation in trans.
  • Example 8 in which the use of lactose minimal growth medium in the bioassay is combined with the use of the Lac repressor as a test DNA sequence which controls the expression of the LacZ component of the beta- galactosidase gene as the reporter gene. lacZ expression is required for host cell growth, and the gene is repressed if non-mutated Lacl is present, thereby selecting for mutation events.
  • An alternative selectable system utilizes a growth advantage provided by the expression of a reporter gene, such that upon mutation of the target gene, the reporter gene is expressed when the target gene is assayed. Thus clones containing mutations on the target gene are "selected" by the growth selection over clones without the mutation.
  • Selectable reporter genes have been described earlier, and include antibiotic resistance, and the groE and S 5 systems described herein.
  • Another selectable system involves the use of a tRNA suppressor gene on the test DNA sequence and the use of a reporter gene which depends of suppression for expression.
  • Still another selectable system involves the vise of anti-terminator proteins to regulate the reporter gene transcription unit.
  • the reporter gene transcription unit contains, in addition to the reporter gene itself, terminator and anti- terminator nucleotide sequences in the promoter region of the transcription unit that terminates transcription unless an anti-terminator protein is present that specifically binds to the anti-terminator sequence.
  • a controlling element in such a system contains both a terminator sequence and an anti-terminator sequence that overrides termination of transcription and is referred to collectively as a terminator/anti-terminator nucleotide sequence. Exemplary terminator/anti-terminator nucleotide sequences are described in Examples 9-11.
  • the target gene system contains, in addition to the test DNA gene sequence, an anti- terminator gene that expresses an anti-terminator protein that specifically binds the anti-terminator sequence in the reporter gene transcription unit and derepresses the terminator sequence, thereby allowing transcription of the reporter gene product.
  • terminator/anti-terminator sequences in the reporter gene transcription unit provides a particular advantage in the present invention.
  • the anti-terminator protein coding sequence on the test DNA sequence that is introduced into the host cell, the anti-terminator protein acts in trans relative to the terminator/anti-terminator sequence, and is not present until the test DNA sequence is added to the host cell bioassay system.
  • the reporter gene transcription unit is kept off until the test DNA sequence is added, thereby reducing and preferably preventing any possible leakiness to the reporter gene, and thus any false regulation of reporter gene transcription.
  • No reporter gene product can accumulate prior to the introduction of the test DNA due to low grade transcription of the reporter gene transcription unit. This insures a low background to the system, and blocks against competing mutations that might independently activate the reporter gene which arise in the host cell rather than in the test DNA sequence.
  • a preferred anti-terminator protein is the lambda N protein that binds to the lambda nutR anti- terminator sequence or the lambda Q protein that binds to the lambda qut anti-terminator sequence.
  • Numerous terminator and anti-terminator sequences are known in the art that are suitable for use in the present invention.
  • the lambda nutR or nutL anti- terminator sequences are regulated by binding by lambda anti-terminator protein N
  • the lambda qut anti-terminator sequence is regulated by binding by lambda anti-terminator protein Q.
  • Terminator sequences that terminate transcription in the absence of anti-terminator protein can include any combination of one or more terminator sequences.
  • terminators include the lambda terminators tl, tLl, tL3, tRl, tR2, t6s (also known as t'Rl), t'Jl, t'J2, t » J3, t'J4 and tRO, the E. coli terminators IS1, IS2 and tryptophan gene terminator (tTRP) , phage P82 tR' (t82), phage P22 tANT, tI4 and tfd.
  • Terminator tRl is particularly preferred and is exemplary of the diversity of numbers and position of terminators possible for use with an anti-terminator sequence.
  • tRl is known to contain 5 terminator sites, designated as tRl(I-V), and various combinations of the 5 sites can be used, such as tRl(I-II), tRl(I-III) and tRl(I-V), as described herein.
  • multiple different terminators can be used, such as to combine tRl with t6s, and the like combinations as described in the Examples. Exemplary systems that utilize a terminator sequence are described in Examples 9, 10 and 11.
  • a number of genetic variables are contemplated which can be modified to optimize the sensitivity of the system that depend, in part, upon the particular phenotype being utilized as the detection of a reporter gene. These variables include (1) the type and thereby strength of the promoters used to express a reporter gene, the (2) the type and thereby the strength of the operators used to control the promoters, and (3) the copy number of the reporter gene transcription unit within the host cell relative to the copy number of the test DNA sequence. It is understood that for any phenotype dependent upon the regulation of gene expression there are optimum, and therefore preferred, combinations of promoters, operators and gene copy number.
  • Promoters for use in controlling a reporter gene include Placl q , lacP, and Ptrp, which each exhibit a different promoter strength, and their selection depends on the promoter strength desired in adjusting the level of transcription for the reporter gene transcription unit.
  • Prolysogenic Organisms The present invention also contemplates a selectable system for screening (testing) for the presence of mutagenic activity upon the target gene system of this invention that utilizes a prolysogenic organism to provide positive selection for mutagenized target genes.
  • the invention also contemplates a prolysogenic organism for use in the system and methods of this invention.
  • a preferred prolysogenic organism is a prolysogenic microorganism and will be used as exemplary herein.
  • a prolysogenic microorganism (prolysogen) is a microorganism containing an isolated bacteriophage cl gene.
  • isolated bacteriophage cl gene is meant a cl gene separated from other bacteriophage genes.
  • the isolated cl gene is present in the microorganism operatively linked to expression control elements for producing a bacteriophage lytic cycle-suppressing amount of cl gene product in the microorganism.
  • the microorganism can be any microorganism, such as a yeast, bacterium and the like, adapted for infection by a bacteriophage, and preferably is a strain of E_-_ coli .
  • a lytic cycle-suppressing amount of cl gene product is an amount sufficient to prevent a lambda bacteriophage-infected cell from lysing during the lytic phase of the bacteriophage's life cycle.
  • the study of bacteriophage lambda is extensive in the biological arts, and the life cycle, and the lytic and lysogenic phases of the lambda life cycle are extremely well characterized.
  • assays for determining whether the cl amount is sufficient for suppression of the lytic cycle, and produce a lysogenic infection is well known in the art.
  • the microorganism expressing a lytic cycle- suppressing amount of cl gene product is referred to as a prolysogen to connote its ability to impose a lysogenic life cycle upon a lambda-infected cell, even if the lambda would otherwise have the ability to be lytic.
  • the control of lytic versus lysogenic life cycles for lambda bacteriophage is well known to reside in the expression of the cl gene product.
  • the prolysogen is phage-free, i.e., is free of genetic material recoverable via a bacteriophage packaging extract.
  • a prolysogen that is restriction system deficient e.g., a prolysogenic strain of E.
  • the prolysogen not contain any restriction system similar to a restriction systems found in the minute 98 region of E. coli K-12.
  • Methods for producing an isolated cl gene are well known in the art.
  • a preferred method utilizes PCR amplification of the gene and its native promoter as a single DNA segment no more than about 1000 nucleotides in length. Typical and preferred are the methods described herein.
  • the cl gene-containing DNA segment is then typically operatively linked to an genomic insertion element, such as a transposon, or the insertion elements of the transposon.
  • the cl gene-containing DNA segment can be linked to a plasmid capable of low copy number maintenance in the host.
  • the amount of cl gene, and therefore the amount of cl gene product expressed can vary, so long as the amount is sufficient to suppress lytic cycle, as described previously. To that end, the number of copies of the cl gene can vary, although typically 1 to 4 copies are preferred, particularly 1 copy as demonstrated herein in the preferred embodiment of the SCS-8cI lysogen.
  • kits for practicing the methods of the present invention that comprise, in an aliquot, a prolysogen of this invention.
  • a kit can further contain a lambda phage packaging extract of this invention for use with the prolysogen, and having a restriction system deficiency compatible with the prolysogen.
  • BHB2690, which is D " are available from the American Type Culture Collection (ATCC) , Rockville, MD under the accession numbers 35131 and 35132, respectively.
  • RecA + transformation is accomplished by standard methods, typically using a RecA expressing plasmid.
  • 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, 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 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 BHB2688McrA " and BHB2690McrA " .
  • Step 9 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.
  • Mrr restriction activity by comparing plating efficiency of lambda versus lambda which has been in vivo methylated by Pst I methylase (Heit an, supra) .
  • An Mrr + strain will show reduced efficiency with the methylated lambda.
  • the Mrr " strain is preferably also McrF " .
  • McrF is tested using packaged lambda DNA that has been in vitro methylated with SssI methylasse.
  • An McrF + strain shows a reduced number of plaques when using methylated lambda DNA.
  • HsdR restriction activity by comparing plating efficiency of lambda versus lambda which has been in vitro methylated by HsdM methylase (Wood, J. Mol. Biol..
  • a bacteriophage PI lysate hereinafter referred to as PI, was made from any E. coli K12 strain that carries a tetracyline resistant Transposon 10 (TnlO) in or near the mcrA gene (TnlO: :McrA) . Briefly, one drop from an overnight culture of K12 was admixed into 5 ml of LB broth (Luria-Bertani broth was prepared by admixing and dissolving 10 grams (g) bacto-tryptone, 5 g bacto-yeast extract and 10 g NaCl into 1 liter deionized water) containing 5 X 10 "3 molar (M) CaCl 2 .
  • the admixture was aerated by swirling until the cells were in exponential log phase growth and had reached a density of 2 X 10 8 cells/ml.
  • 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.
  • BHB2690 (ATCC # 35132) was used as the specific strain for transduction.
  • E. coli BHB2690 which was RecA ' , as 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.
  • 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) .
  • McrA " strains were then purified and tested for the lack of McrA restriction activity. The determination of McrA " strains was accomplished by comparing transformation efficiency of unmethylated pBR322 versus pBR322 that had been in vitro methylated by Hpa II methylase. A McrA " strain showed a greatly reduced efficiency with the methylated plasmid. BHB2690RecA + McrA " ; hereinafter designated BHB2690McrA " , strains were, thus, determined and used to make BHB2690McrB " transductions as described below.
  • 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 R 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.
  • the Mrr- strain is preferably also McrF " , as described before.
  • 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. With these tests, a selected colony which lacks all restriction activity, McrA, McrBC, McrF, Mrr, and HsdR, and constructed using this transduction approach was shown to contain a deletion throughout the McrB region. The resulting strain was grown in liquid culture and then plated to isolate colonies on NZY media.
  • E. coli BHB2688 strains containing RecA* but lacking McrA, McrBC, McrF, 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.
  • E. coli lysogen, strain BHB2690R- (prehead donor) prepared in Example la the genotype of the strain is first verified before large-scale culturing.
  • the presence of the mutation that renders the bacteriophage cl gene product temperature-sensitive is determined by streaking from the master stocks of E. coli BHB2690R- onto two LB agar plates. One of the plates is maintained at 32°C while the other is maintained at 45°C. Bacteria with intact cl only grow on the plates maintained at 32 °C. A single small colony of E. 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.
  • NZM broth is prepared by admixing 10 g NZ amine, 5 g NaCl, and 2 g MgS0 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 OD 600 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 600 of approximately 0.3 is reached.
  • the OD 600 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.
  • the flask 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 M Tris- HC1, pH 8.0, (Tris[hydroxymethy1]-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 induced E. coli BHB2688R- cells are pelleted by centrifugation at 4000g for 10 minutes at 4°C. The resultant supernatant is removed and any excess liquid is removed. 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 packaging extracts can be admixed with the packaging reaction either simultaneously or by multiple additions of the packaging extract.
  • a comparison between the single addition and multiple addition of extracts to packaging reaction admixture was made as follows. A conventional procedure using 4 ul of DNA to be packaged was admixed with 10 ul of freeze-thaw extract and 15 ul of sonicate extract, and the admixture maintained over 3 hours at 30°C. Thereafter, the titer of phage packaged was determined. In contrast, 4 ul of DNA was admixed with 5 ul of freeze-thaw extract and 7 ul of sonicate extract and maintained for 1.5 hours at 30°C.
  • 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.
  • a variation of L2B was constructed that contains a plasmid sequence that can be readily excised from the lambda phage and contains the lacl repressor gene.
  • This variation has several advantages. First, as discussed below, physical identification of phage carrying mutations is facilitated since they grow as blue plaques on a white background in the presence of X-gal (5-bromo-4-chloro- 3-indolyl-3-D-galactopyranoside) without IPTG (isopropyl/3-D-thiogalacto-pyranoside) . This advantage also simplifies and reduces the cost of the assay since it permits an increase in the density of phage per plate. Additionally, the lacl genetic systems of E.
  • coli are the first systems that conveniently permitted the study of large numbers of mutations within procaryotes at the DNA level (Miller et al, J. Mol. Biol., 109:275-302 (1977), Coulondre et al, ___ Mol. Biol., 117:275-302 (1977), Schaaper, J. Mol. Biol. , 189:273-284, (1986)), and the use of lacl provides a test gene with significant historical mutational data for comparison between mutagenesis assays.
  • mice were used as the test animal. (Hogan et al, Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986) . Single cell mouse embryos were harvested from female mice that were impregnated the evening before. The embryos were treated with hyaluronidase and briefly cultured in M16 medium. The embryos were transferred to M2 medium on a microscope glass depression slide. The embryos were observed with a 4OX objective and a 10X eyepiece using a Nikon Diaphot microscope equipped with Hoffman optics. The embryos were held in place with a holding pipet that had been rounded with a microforge. The positions of both the holding pipets and the injection pipets were controlled with icromanipulators.
  • 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.
  • 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.
  • the above procedure has been utilized with the L2B vector as the source of test DNA sequence, and has also been utilized with other test DNA sequences, including lambda LIZ Alpha, described herein.
  • the resulting transgenic mouse containing the test DNA sequence lambda LIZ alpha was deposited with the ATCC in the form of a frozen mouse embryo as described further herein.
  • mice Six to eight week old transgenic male mice were treated on day 1 and day 4 by intraperitoneal injection of either 125 or 250 mg N-ethyl-N- nitrosourea (EtNu) , per kg body weight. Control animals were injected with 100 mM phosphate buffer at 10 ml/kg body weight. Tissues were collected two hours after final injection.
  • EtNu N-ethyl-N- nitrosourea
  • rescue of the marker (test) 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.
  • 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 plaque forming 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 packaged 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°, 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 S gene that is required for cell lysis. 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, McrBC, McrF, HsdR 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 “ , McrBC “ , McrF “ , Mrr “ , HsdR “ , and preferably prepared from K-12 * mcrB region, 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. (2) Testing for Mutagenesis
  • 3-galactosidase deficient E. coli are grown in IX TB (5g/L NaCL, lOg/L tryptone) supplemented with 0.2% maltose and lOmM MgS0 4 overnight at 30° C. Cells are harvested by centrifugation and resuspended in lOmM MgS0 4 in preparation for plating (Maniatis, supra) .
  • 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 fr me, altered essential codons, or has created stop codons in the sequence.
  • the ratio of white or faint blue to blue plaques minus background provides a numerical measure of the mutagenic potential of the mutagen being tested. Background is the measured mutation rate 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 et al, Proc. Natl.
  • fibroblast cell lines are obtained from organisms containing the test DNA sequence of interest. Adams, 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.
  • the Hsd system works by selectively restricting DNA that is not protected by adenine methylation at the N-6 position in the sequence, A ⁇ ACNNNNNNGTGC or GC 6me -ACNNNNNNGTT.
  • 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 mrr gene system has recently been shown to also recognize and restrict cytosine methylated sequences. This activity of the mrr gene has been named McrF.
  • McrA and McrBC are similar to Mrr in that they recognize and restrict methylated DNA. However, these two systems differ from Mrr in that they have been shown to recognize only methylated cytosine.
  • McrB function is provided by the products of at least two genes, mcrB and mcrC (Ross et al, J. Bacteriol.. 171:1974- 1981 (1989)) .
  • the recognition sequences for mcr and mrr are contemplated in the literature, but precise sequences are as yet unknown.
  • Strain RR1-A and K-12*mcrB are constructed as described below. Strain RR1-A is constructed with strain RR1
  • Step 1 A PI lysate is made from the E. coli K-12 strain described above.
  • Step 2 RR1 is transduced (Miller, J. , Experiments in Molecular Genetics, Cold Spring Harbor Lab., Cold Spring Harbor, New York (1972)) .
  • 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.
  • 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. coli K-12 strain that carries a TnlO(tet R ) in the mcrB gene.
  • 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* 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 vitro methylated by Hsd methylase (Wood, 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 activities, namely, McrA, McrBC, McrF, Mrr, HsdR and was constructed by this method, it should then contain a deletion throughout the McrB region (AmcrB) . It will then also very efficiently plate lambda that has been rescued from the mouse. This strain is called K-12AmcrB. 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 1 are K-12 derivatives believed to contain a small mutation in the mcrB region, with the exception of E.
  • a "+" plating efficiency of phage indicates that approximately 500 pfu/0.05 ⁇ g of transgenic mouse genome DNA was observed, while a “-" plating efficiency indicates that less than 5 pfu/0.05 ⁇ g of transgenic mouse genome DNA was observed. Note also that (+) indicates that the Mrr activity has not been confirmed in Y1088.
  • the 98 minute region of E. coli K-12 LCK8 was cloned.
  • a partial LCK8 genomic library was made in pOU61cos. (Knott, Nucleic Acid Res. , 16:2601-2612 (1988)) , packaged with GigapackTM II XL
  • a complete deletion of the minute 98 mcrB region is preferred, as opposed to a small mutation of mcrB present in most commonly used McrB " lab strains. This is because despite the McrB " phenotype exhibited by these McrB " strains (using Alul methylase modified pBR322 transformation as the assay (Ross, supra. ) ) some inhibitory activity of the mcrB region remains. Complete deletion resulted in optimal efficiency, accounting for a greater than 1000-fold improvement in rescue efficiency using eukaryotic modified DNA.
  • 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 kit (Big BlueTM Mouse Mutagenesis System, Catalog Number 720,000).
  • SCS-8 has the following genotype: RecAl, endAl, mcrA, (mcrBC-msdRMS-mrr) , ⁇ * ⁇ (argF- lac)U169, phi80 lacZAM15, Tnl0(tet r ) .
  • SCS-8 provides the lacZAM15 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
  • 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.
  • 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.
  • the target lambda phage can be made to provide a target gene with reduced size (e.g., the lacl 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 lacl and ⁇ -gal genes are inserted within a lambda vector, such that if the mutation occurs within the lacl gene, the repressor activity is lost allowing the ⁇ -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 et al. The Operon, 2nd Ed.
  • the AM15 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.
  • 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 t>y 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(-) , lacZAM15, restriction(-) .
  • PR' which prevents lacZ expression in the host E. 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 et al, Nucleic Acids Res. , 16:7583-7600 (1988) . pMJR1560 is available from Amersham Corp., Arlington Heights, Illinois. Rapid sequencing of the mutagenized lad gene within the lambda vector is facilitated by incorporating "lambda ZAP" excision sequence within the lambda vector. (Short et al, Nucleic Acids Res.
  • 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 lad 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 et al, Proc. Natl. Acad. Sci. USA, 82:7270- 7274 (1985) ; Jayaram, Proc. Natl. Acad. Sci. USA, 82:5875-5879 (1985); McLeod, Mol. Cell.
  • 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 thy idine 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.
  • antibiotic resistance gene sequence ampicillin, kanamycin, tetracycline, neomycin, chloramphenicol, etc.
  • the lambda Red and Gam gene sequences a thy idine kinase gene sequence
  • a xanthine-guanine phosphoribosyl transferase gene sequence sequence
  • test sequence(s) By bracketing the test (marker) 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 (enzyme rX) and subsequently ligated with restriction sites of 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.
  • restriction enzymes enzymes
  • Lambda LIZ alpha An exemplary 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 MI-.
  • pBluescript SK- (Stratagene, La Jolla, California) is modified using site directed mutagenesis to introduce an Avalll 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-.
  • Figure 5 depicts the construction of pLadq.
  • pBluescript II SK+ (Stratagene) is digested with the restriction enzymes Pstl 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 Pstl and EcoRI to release a lacl q - containing fragment that is separated by agarose gel electrophoresis and eluted from the gel. The lacl q - containing fragment is then ligated into the linearized pBluescript SK+ to form pLa q.
  • Figure 6 depicts the construction of plnt.l.
  • a double stranded DNA segment defining multiple cloning sites (a polylinker) is produced by synthetic oligonucleotide synthesis and annealing.
  • the polylinker contains multiple restriction endonuclease recognition sequences including two Avalll sites flanking Xbal, Kpnl and Pvul sites.
  • the polylinker is digested with Avalll to form Avalll cohesive termini on the polylinker.
  • pBlueMI- is digested with Avalll and the polylinker is ligated into pBlueMI-to introduce the Pvul site into the Avalll site and form pint.1
  • Figure 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 #37254) described by Yanisch-Perron et al, Gene, 33:103-119 (1985) , was digested with EcoRI, dephosphorylated and ligated to complementary oligonucleotides, each having compatible EcoRI ends and defining a T7 RNA polymerase promoter as described by McAllister et al, Nucl. Acids Res. , 8:4821-4837 (1980) to form pJF3 having the T7 promoter oriented to direct RNA synthesis towards the multiple cloning site of pUC 19.
  • 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.
  • Phage id 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.
  • telomere sequences were provided by preparing synthetic oligonucleotides as described by Short et al, supra, to provide a complete terminator, a gene II cleavage signal and unique restriction sites for EcoRV and Ndel.
  • 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 pBSIT0#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 Sail and the resulting small fragment containing the lacl q gene was isolated.
  • 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 plntladqZ.
  • plntladqZ was then digested with Xbal, blunt-ended with Klenow, digested with Seal, and the resulting large fragment containing lacZ-lad q and most of the a picillin 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 (srIlambdal-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 1 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 lacl q target gene, including a lad promoter and the repressor structural gene sequences, and 4) an fl origin of replication arranged according to the present invention and as described for 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
  • PBS phosphate buffered saline
  • Lymphocytes are also isolated from tonsils obtained from therapeutic tonsillecto ies from consenting patients. The tonsils are first homogenized and then lymphocytes are isolated over Histopaque as described above.
  • Subject rDNA are then inserted into the isolated lymphocytes using techniques known to one skilled in the art. Preferred techniques are electroporation of lymphocytes and calcium chloride permeabilization of the lymphocytes.
  • 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).
  • Analysis of the pedigree of 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.
  • 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.
  • a prolysogenic microorganism (a prolysogen) was prepared as described below, and was used as exemplary of the methods of this invention.
  • the prolysogen was constructed in E. coli strain SCS-8 available from Stratagene (La Jolla, California) using the procedures of Herrero et al, J. Bacterial. , 172:6557-6567 (1990), to introduce a stably integrated copy of the lambda cl gene into the genome, and form the E. coli designated SCS-8cI.
  • the genotype of E. coli strain SCS-8 has been described in some detail by Kohler et al, Proc.Natl.Acad.Sci.USA, 88:7958-7962 (1991).
  • SCS-8 is resistant to the antibiotic tetracycline.
  • the prolysogen E. coli strain SCS-8cI has been deposited with the American Tissue Culture Collection (ATCC; Rockville, MD) on February 14, 1992, and has been assigned an ATCC accession number 55297.
  • pUC18Sfi I was first prepared as described by Herrero et al, supra.
  • pUC18Sfi I was derived from pUC18 by adding two Sfil restriction sites flanking the polylinker.
  • Two oligonucleotide primers were synthesized that correspond to the termini of the wild type cl gene nucleotide sequence described in "Lambda II" by Hendrix et al, at page 631. These two primers (pi and p2) were designed for use as PCR primers to amplify a cl gene-containing fragment when used on wild type lambda in he form of a E. coli lysogen extract of strain N.
  • the primers have the nucleotide sequences as follows: pi 5 ⁇ -ATCAGCGAATTCCAACCTCCTTAGTACATGCAA-3 ' , and p2 5'-CATACGGTCGACGATCAGCCAAACGTCTCTTCAGG-3 ' .
  • pi SEQ ID NO 5
  • p2 SEQ ID NO 6
  • the resulting PCR product was inserted into the polylinker region of the plasmid pUCl ⁇ Sfi I, and then removed from the plasmid by digestion with Sfil, to form a PCR fragment having Sfil cohesive termini.
  • pUTKm contains the 19 base pair ends of the transposon Tn5 required for insertion of DNA fragments into genomic DNA. Between the flanking ends, termed insertion elements, is a selectable marker, the gene for kanamycin resistance (kan) , and a restriction endonuclease site for Sfil into which the PCR fragment containing the cl gene was ligated.
  • the tnp gene encoding the transposase protein required for the transposition function.
  • the tnp gene is left behind, so that the genomically integrated insertion fragment having the cl and kan genes cannot be excised by transposition in the absence of a transposase protein.
  • Plasmid pUTKm also contains an R6K origin of replication (ori) that requires the pir gene product for replication.
  • pUTKm was propagated in the S17-1 E. coli strain that contains the pir gene (pir + ) also described and provided by Herrero et al, supra.
  • S17-1 is sensitive to both of the antibiotics kanamycin and tetracycline.
  • Plasmid pUTKm-cI was transformed into E. coli S17-1 according to standard bacterial transformation methods, and cultured in kanamycin to select for transformants containing the plasmid, and were designated sl7-l/pUTKm-d.
  • the absence of the pir gene product in the SCS-8 cells prevents the plasmid pUTKm-cI from replicating, thereby providing the selective pressure for transposition to occur in order to maintain the kanamycin gene in the E. coli cells.
  • the resulting viable cells are SCS-8 E. coli containing integrated cl by transposition, and having antibiotic resistance to both kanamycin and tetracycline at 50 and 15 ug/ml, respectively.
  • SCS-8cI cells are designated SCS-8cI cells, and are exemplary herein as a prolysogenic microorganism because the expression of the cl gene product prevents the cell from entering the lytic phase upon infection by a lysis-competent lambda bacteriophage when cultured at 30°C as described in Example 8.
  • the lac repressor When a "phenotypic" mutation was present in the lad gene, for example, the lac repressor is no longer able to block expression of the alpha-lacZ gene that is present in the recovered phage genome.
  • This alpha- lacZ protein is then able to complement with the o ega-lacZ protein that is produced constitutively in the host E. coli strain to form a functional beta- galactosidase protein, referred to as alpha- complementing beta-galactosidase.
  • This protein then breaks down the chromogenic substrate, X-gal, that is present in the top agar media of the plate.
  • X-gal chromogenic substrate
  • both mutant and non-mutant targets are scored in this assay, it is considered a non- selectable system for screening for mutagenesis of the target gene.
  • this system generated easily identifiable mutants, plating densities cannot exceed 50,000 plaques per 25x25 cm plate and are optimal at densities of 15,000 plaques per plate. Based on these numbers, 10-20 plates are required for each mouse tissue analyzed. The number of plates contributes significantly to the cost of the assay in terms of plates, media, X-gal substrate and labor.
  • the system described in this example is a selectable, and preferred, version of this assay in which only phage that harbor mutant lad target genes can survive and be identified on the plate. This selection allows a higher plating density to be used: up to 500,000 phage and thus 500,000 target genes, can be screened on one 25x25 cm plate, significantly decreasing the cost and time to perform the assay.
  • mutants using the present selectable system depends on the expression of the alpha-JLacZ gene to create a functional beta- galactosidase protein.
  • This protein allows the cell to utilize lactose for growth.
  • cells carrying phenotypically mutant lad genes can grow on minimal media containing lactose as the sole carbon source.
  • Cells carrying non-mutant lad genes code for a functional lac repressor and therefore cannot express alpha-lacZ, and die from carbon starvation.
  • the system is selectable for only those target genes which have been mutagenized, thereby inactivating the lacl gene.
  • the plating media required for this selection is referred to as minimal media containing lactose as the sole carbon source and consists of the following components expressed per liter and are obtained from Sigma Chemical Co. (St. Louis, MO) unless noted differently: 6.0 g Na 2 HP0 4 , 1.0 g NH 4 C1, 0.5 g NaCl, 20.0 g Bacto Agar, 0.5 g lactose, 0.34 g thiamine HCl, 1.0 ml of IM Mgs0 4 , 0.1 ml of IM CaCl 2 .
  • the top agar consists of the following components expressed per liter: 6.0 g Na 2 HP0 4 , 3.0 g KH 2 P0 4 , 1.0 g NH 4 Cl, 0.5 g NaCl, 3.5 g Seakem Agarose (FMC, Rockland, ME), 0.4 g Difco casamino acids, 2.0 g X-gal (Stratagene Cloning Systems, La Jolla, CA) . Note that X-gal was used in this media not to allow distinction between mutants and non-mutants but to allow easier identification of the mutants on the light colored minimal media plates.
  • Variations in the media formulation can be utilized, however, it was determined that lactose should not be included in the top agar, as this contributed to elevated levels of false positive bacterial cell growth (higher backgrounds) . In addition, the rate of growth for the lactose-dependent cells was improved when casamino acids were included in the top agar. In addition to the change in media that is required for the selectable system to function, a new E. coli strain was also required. Because the selection depends on survival of the host cells as opposed to plaque formation, it is necessary to inhibit lytic growth of the rescued phage particles once they have adsorbed to the host E. coli cells. This was done by using a strain of E.
  • coli that carries the lambda cl gene, which strain being referred to herein as a prolysogenic strain because it maintains the lambda infection in a non-lytic life cycle.
  • the cl gene specifies the lambda repressor protein which allows lambda genomes to be maintained in the lysogenic state as opposed to replicating lytically.
  • the cl gene is stably integrated in the E. coli chromosome as described above.
  • E. coli strain SCS-8cI and the minimal media described above are the two major changes that are incorporated into the protocol described in the earlier Examples 1-6 for the non-selectable version of mutagenesis testing assay of this invention.
  • the general method of the assay was as follows: First, genomic DNA was isolated from transgenic mouse tissue. Approximately 20.0 ul of the DNA was then packaged using Transpack in vitro lambda packaging extract (produced as described earlier) for three hours at 30°C. SM buffer was then added to the reaction tube to give a final volume of one ml. Fifty ul of the reaction was then plated according to the protocol described for the previous non-selectable system.
  • the remaining 900 ul of the packaging reaction was then adsorbed to SCS-8cI cells (prepared as previously described with the exception that the cells are resuspended to an OD of 2.0 after spinning), for 20 minutes at 30°C followed by mixing with 2.5 ml of minimal top agar and pouring onto a 25 x 25 cm plate containing lactose minimal media. This plate was then incubated for approximately 60 hours at 30"C before scoring the blue mutant colonies. The non-selectable plate was scored for total number of plaques to determine the rescue efficiency as previously described. Note that in this system one packaging reaction was used to set-up two plates: one non- selectable plate that allows determination of rescue efficiency and one selectable plate that allows
  • this selectable system allows very high rates of lysogenization.
  • the data also shows that cells carrying a lysogenized phage with a mutant lad gene are able to form colonies on this media while those cells carrying phage with non- mutant lad genes are not able to form colonies.
  • the lysogenization/selection system functions extremely efficiently.
  • the selectable system was then evaluated for its ability to detect both spontaneous and induced mutant lad targets rescued from the mouse genome.
  • genomic DNA was prepared from the liver of animals treated as described in previous assays, as well as from untreated control animals. Briefly, 6-8 week old B6C3F1 mice were injected intraperitoneally with 100 mg MNU/kg/day for five days. The animals were sacrificed 12 days after the injections, and the genomic DNA prepared and assayed as described. The selectable mouse mutagenesis assay was then performed on three separate days (experiments 1, 2, 3) as described above.
  • the results are shown in Figure 9 as mutant frequency versus time.
  • the mutation frequency is the number of mutant colonies obtained divided by the total number of phage particles screened as calculated from the non-selectable plate.
  • the time represents the hours at which the mutant colonies were counted after being placed in the 30°C incubator.
  • the data show that both spontaneous and induced mutant lad targets can be detected with this selectable assay allowing the calculation of mutant frequencies.
  • the data also demonstrates the reproducibility of the assay as determined by three independent experiments. Additionally, induction rates can be detected with this system as seen by the increased number of mutants observed with the treated (induced) DNA relative to the untreated (control) DNA.
  • the data in Figure 9 is expressed over time because mutants continue to arise over time. It is possible that a correlation exists between the time that a mutant colony appears and the specific type of mutation that is present in the lad gene. This may aid in classifying the mutants. For example, a colony that appears early may contain a "strong" mutation while a colony that appears late may contain a "weak” mutation that confers more of a "leaky” lacZ phenotype.
  • the selectable system described above is flexible in that the components and concentrations of the components of the minimal media may be varied as can the incubation temperature of the plates. These changes can effect the growth rate of the cells and thus alter the time at which the plates can be scored.
  • the selectable version of the previously described mouse mutagenesis assay can be used to determine mutation frequencies and induction rates.
  • two major modifications to the original assay were required: the new E. coli strain (designated as a prolysogen) was constructed, and a different plating media was developed.
  • This system allows ⁇ 10 ⁇ 20 fewer plates to be used, thus significantly reducing the cost of the assay in terms of plates, media, X-gal chromogenic substrate, and labor.
  • the selectable system allows the classification of lad mutants and permits a wider and/or different spectrum of mutants to be detected.
  • the above selectable system utilizing the SCS-8cI host cell and minimal lactose medium was conducted as above but at 37°C, rather than at 30°C, in order to reduce the growth time. Under these conditions, although lysogenization was induced, the lac repressor was unable to sustain lysogeny at 37°C for prolonged periods, and plaques form in the minimal media top agar after about 40 hours.
  • the casamino acids in the top agar permitted the growth of a low density lawn of E. coli necessary for the plaque formation by the phage harboring the mutant lad gene. Without the supplement, plaques were not visualized.
  • the present embodiment allows mutants to be scored as lytic plaques after shorter incubation times of about 40 hours, rather than the longer 60-65 hours required when growth is at 30°C with the lysogenic assay. 9. Construction of a Gene Activating Selectable
  • a plasmid vector containing a reporter gene under the transcriptional control of a lac promoter and lac operator is constructed having the E. coli groE as the reporter gene and the lambda terminator nucleotide sequences qut-t6s that is regulated by lambda terminator protein Q.
  • a strain of E. coli that is GroE " is required as the host cell for use of the reporter gene construct.
  • E. coli Strain SCS-8 tet r described earlier was selected for sensitivity to tetracycline by culturing on Bochner media as described by Maloy et al, J. Bacteriol. 145:1110-1112 (1981) to form SCS-8 tet s .
  • the strain SCS-8 tet s was transformed with the RecA + plasmid pJC859 described earlier herein, to form SCS-8 tet s /pJC859.
  • a PI lysate was prepared according to standard procedures from E. coli strain CAG9269 described by Tilly et al, Proc. Natl. Acad. Sci USA.
  • E. coli strain SCS-8 tet r groEL140/pJC859 was cultured under conditions of rich liquid media (NZY) culture to select for the loss of pJC859 to form SCS-8 groEL140 (tet°, which has the relevant genotype of groEL140, recA, deltaM151acZ, mcrA, delta(mcrCB-hsdR- rr) Sup 0 .
  • the resulting GroE mutant E. coli strain is extremely efficient at inhibiting lambda plaque formation, and no plaques are observed when 4.5 x 10 8 phage particles are plated on strain SCS-8 groEL140 (tet r ) . This level of inhibition is well below the amount of spontaneous mutations of about 10 "5 required for use in a mutagenic testing animal of this invention.
  • a second E. coli strain designated SCS-8 groES30 (tet r) was similarly prepared using the E. coli strain CAG759 described by Fayet et al, J. Bacteriol. , 171:1379-1385, (1989), that is groES30 TnlO tet r as the source of the PI lysate.
  • groE gene was PCR-amplified from a colony of E. coli strain XLl-Blue (Stratagene) using Taq DNA polymerase under standard PCR procedures with the following primers p3 (SEQ ID NO 7) and p4 (SEQ ID NO 8) : p3 5'-GCCGGTCGACCTAGTAAGGACTTTCTCAAAGGAGAGT-3' p4 5*-GTCAGGGCCCCGTGCATGTTATTCCCCATA-3 '
  • the resulting PCR-a plified fragment contains a ribosome binding site, an ATG translational start sequence, and both the groES and groEL genes.
  • a groE gene can readily be isolated from other E. coli strains. Thereafter, the fragment was restriction endonuclease digested with Apal and Sail, treated with calf intestine alkaline phosphatase (CIAP) , gel purified and cloned into pBluescript SK (pBS; Stratagene) that had been pre-cut with Apal and Sail to form pBSG3.
  • Plasmid pBSG3 contained the groES and groEL genes under the control of the lac promoter and lac operator in pBS. Plasmid pBSG3 is designated a high copy number plasmid because it contains a ColEl prokaryotic origin of replication that maintains the plasmid in the host cell at high copy number. Thus host cells used in the assay system of the present invention will contain high copy numbers of plasmid where the reporter gene transcription unit is based on pBSG3.
  • Plasmid pl5 SK was provided by Ronald Fisher (Federal Republic of Germany) . Plasmid pl5 SK contains the pl5A replicon and is based on the pBluescript SK plasmid except for the replicon, and the presence of the chloramphenicol acetyltransferase gene (cat) selectable marker.
  • Plasmid pBSG3 was digested with Apal and Sail to release the groE gene, and the resulting digested plasmid treated with CIAP.
  • the resulting 2.2 kilobase (kb) fragment containing the groE gene was isolated, purified, and cloned into pl5 SK that was predigested with Apal and Sail to form plasmid pl5G10.
  • Plasmids pl5G10 and pBSG3 were tested for their ability to complement GroE mutant E. coli strain CAG9269 groEL140.
  • each plasmid was transformed into the mutant strain to form CAG9269 groEL140/pl5G10 and CAG9269 groEL140/pBSG3.
  • About 280 particles of lambda L2B were plated on XLl-Blue, on CAG9269 groEL140, on CAG9269 groEL140/pl5G10 and on CAG9269 groEL140/pBSG3 , to yield 288, 0, 260 and 161 plaques, respectively.
  • Lambda LIZ Alpha were plated on XLl-Blue, on CAG9269 groEL140, on CAG9269 groEL140/pl5G10 and on CAG9269 groEL140/pBSG3 , to yield 203, 0, 212 and 0 plaques, respectively.
  • Terminator sequences are cloned and inserted into the reporter gene transcription unit of the above described groE plasmids pBSG3 and pl5G10.
  • a DNA fragment containing the lambda terminator sequence qut-t6s was isolated from wildtype lambda by PCR amplification using the primers p5 (SEQ ID NO 9) and p6 (SEQ ID NO 10) : p5 5 '-ATCAGCCTGCAGGTCGACATGGGTTAATTCGCTCGTTGT-3 ' p6 5 '-AGCATCGTCGACGGATCCTCTTACCTGTTGTGCAGATAT-3 '
  • the resulting PCR-amplified fragment contains the lambda terminator sequence qut-t6s.
  • the fragment was restriction endonuclease digested with BamHI and Sail, treated with CIAP, gel purified and cloned into pDR720 (BRL; Bethesda, MD) that had been pre-cut with the BamHl and Sail to form pDR720Qut.
  • the construction placed the qut-t6s termination sequence upstream of a galactosidase (galK) reporter gene in the vector, which can be utilized to determine if termination of transcription is being regulated by the lambda anti-terminator protein Q.
  • galK galactosidase
  • a galK mutant strain of E. coli HB101 was grown on MacConkey galactose color indicator agar plates, and forms white colonies indicating that no galK gene product is formed. Cells that utilize galactose on MacConkey plates form pink colonies while cells that do not utilize galactose form white or clear colonies.
  • HB101 was transformed with plasmid pDR720, that expressed the galK gene product, pink colonies were formed, indicating that the pDR720 transcription unit was expressing the galK gene.
  • HB101 was transformed with plasmid pDR720Qut, that contained the termination sequence qut-ts6 in the galK transcription unit, white colonies were observed, indicating that the termination sequence prevented transcription of the reporter galK gene.
  • N99 lambda + is a lambda lysogen that expresses the lambda anti- terminator Q protein at a low level, and that lacks the galK gene.
  • N99 lambda* forms white colonies, indicating that no galK gene product- is expressed.
  • N99 lambda "1" is transformed with pDR720Qut, pink colonies are formed, indicating that the galK reporter gene transcription unit normally terminated by the qut-t6s sequence is expressed because the Q protein in the host cell is able to anti-terminate (i.e. activate) the terminated galK reporter gene transcription unit.
  • the lambda qut-t6s termination sequence is effective at terminating the expression of a reporter gene transcription unit, and that the expression of the lambda Q anti-terminator protein is effective in trans to control transcription of the terminated reporter gene.
  • the cloned lambda terminator sequence qut-t6s was then inserted into the groE reporter gene transcription unit of both the high and the low copy number plasmids pBSG3 and pl5G10 described above.
  • pBSG3 was digested with EcoRI and Spel, phosphatased with CIAP, and the resulting linear vector containing the groE reporter gene transcription unit was gel purified.
  • the terminator sequence was isolated from pDR720Qut by PCR amplification using the following oligonucleotide primers pl3 (SEQ ID NO 19) and pl4 (SEQ ID NO 20) : pl3 5 '-CGGATCAGCTCTAGAACTAGTATGGGTTAATTCGCTCGTTGT-3 ' pl4 5 ' -GCGAGCATCAAGCTTGAATTCTCTTACCTGTTGTGCAGATAT-3 '
  • the resulting PCR amplified product was digested with EcoRI and Spel, and gel purified. Thereafter, the fragment containing the terminator sequence was cloned into the EcoRI/ Spel site of pBSG3 to form pBSG3Qut.
  • a similar construction was prepared using pl5G10, adding the terminator sequence to form pl5G10Qut. Plasmid pl5G10 was digested with EcoRI and Spel as for pBSG3 , and the qut-t6s terminator sequence was inserted as before.
  • the qut- t6s terminator sequence was placed inside the groE reporter gene transcription unit upstream of the structural gene, and downstream of the lac promoter, thereby rendering transcription susceptible to regulation by the anti-termination protein Q.
  • the in trans regulation of the groE reporter gene product by the Q protein is illustrated in Figure 10, showing the use of the test DNA sequence Lambda LIZ Alpha, which contains the lad test DNA sequence and the lambda Q anti-terminator protein.
  • the reporter gene transcription unit illustrated in Figure 10 shows the promotion of the groE reporter gene by a lac promoter/operator in which the lad repressor also controls the transcription of the reporter gene.
  • the presence of the terminator sequence in the reporter gene transcription insures that reporter gene transcription is inhibited in the absence of a test DNA sequence, thereby increasing the sensitivity of the assay.
  • the invention describes a system related to the anti-terminator system described in Example 9, but which includes a dominant negative inactivator gene as the reporter gene (lambda S 5 mutant) and which includes the use of competing transcription as the means for controlling expression of the reporter gene.
  • the system is illustrated in Figure 11, and is referred to as a reporter gene inactivating system in which plaque formation is inhibited until a mutated lad test DNA sequence is introduced that inactivates expression of the reporter gene.
  • Lambda S 5 is a mutant protein of the multi- subunit membrane pore complex that lambda forms in the inner membrane of an E. coli host cell that is SupF in order for lysis of the host cell during a lytic bacteriophage cycle.
  • the complete membrane pore complex is required for a functional pore to be formed.
  • the test DNA sequence e.g., Lambda LIZ Alpha
  • the S 5 mutation is referred to as a dominant negative inactivator gene, because its phenotype is dominant, not recessive, (i.e., defective even in the presence of wild type S protein) and inactivator because the effect is to inactivate plaque formation.
  • Other examples of lambda genes suitable as dominant negative inactivator genes for use in a gene inactivating system include any lambda gene in which multiple protein subunits are required for a functional protein complex such that the presence of a single mutant protein in the complex disrupts the protein complex's function.
  • the lac repressor lacl d" gene is an example of another dominant negative inactivator gene suitable for use in the present gene inactivating system.
  • the lacl d" gene is described by Miller et al., in "The Operon", 2nd edition, Cold Spring Harbor Laboratory, 1980.
  • Competing transcription is also required in the gene inactivating system shown in Figure 11.
  • the system utilizes two transcriptional promoters of unequal transcriptional promotion strength that compete for transcription in converging orientations across the same reporter gene, the weaker promoter (PR 1 ) producing a transcript that yields reporter gene product, and the stronger promoter (TacP) producing a transcript in the wrong orientation for the reporter gene, thereby blocking expression of reporter gene product.
  • PR 1 weaker promoter
  • TacP stronger promoter
  • the gene inactivating system produces a detectable signal in the form of lytic plaque formation when mutated test DNA sequences are introduced because the mutated lad gene cannot repress the competing TacP transcriptional promoter which is controlled by a lac operator, thus the competing transcript overpowers the weaker PR 1 promoter, the mutant S 5 reporter gene is inactivated, no S 5 protein is made, and lytic plaque formation is no longer inhibited.
  • the weaker promoter transcribes the S 5 reporter gene which inhibits plaque formation.
  • the gene inactivating system shown in Figure 11 preferably contains, but does not require, the in trans terminator/anti-terminator system (Q protein on the test DNA sequence and qut-t6s in the weaker reporter gene transcriptional unit) .
  • the terminator/anti-terminator system ensures that no S 5 protein can be produced in the host cell until the test DNA sequence is introduced into the host cell.
  • the construction of a host plasmid vector containing the reporter gene inactivating system is as follows.
  • Lambda ZA5 (Stratagene) contains the genotype PR'-qut-t6s-S 5 on a section of the lambda genome in the order indicated and as required in the reporter gene transcription unit.
  • p7 SEQ ID NO 11
  • p8 SEQ ID NO 12
  • the resulting PCR-amplified fragment was digested with Nhel and Bglll, gel purified and ligated into pBlueMI-, described in Example 5, which had been predigested with Nhel and Bglll.
  • the resulting plasmid containing PR--qut-t6s-S 5 is designated pMI/S 5 .
  • the nucleotide sequence of the lambda S gene is mutated at two positions to form the S 5 mutation as described herein.
  • the nucleotide sequence of the S 5 gene is shown in SEQ ID NO 18.
  • the other lambda sequences defining PR'-qut-t6s in plasmid pMI/S 5 have wild-type sequences and therefore can be readily prepared from a variety of sources of lambda DNA. If needed, the S 5 mutation can be introduced into wild- type lambda using well known mutagenesis procedures.
  • the S 5 mutation in the lambda S gene requires that the E. coli host cells in which inhibition of plaque formation occurs must harbor the SupF mutation, and when used in this system is referred to as the lambda SY5 system.
  • the genotype of a host cell for the S 5 system is restriction minus, RecA " , supF, and preferably lacZdeltaMIS, although the lacZ mutation is not required unless a color selection is desired in addition to the growth selection.
  • E. coli strain LE392.23 which is SupE
  • SupF was made SupF only by using a PI transduction lysate as before prepared from E. coli strain CAG1E7 having a wild type SupE/TnlO. The transposition corrected the SupE mutation.
  • the resulting E. coli was grown on tetracycline to insure stabilization of the transduced supE gene.
  • E. coli strain SCS-15 SupF is rendered restriction minus using the procedures described in Example 4.E.2. Thereafter, the strain is rendered Rec A- by subjecting it to a standard PI transduction using a lysate from an E. coli which contained a TnlO transposon near a mutant recA gene.
  • the plasmid pMI/S 5 was then transformed into E_j_ coli strain SCS-15 (supF) , and tested for inhibition of lytic plaque formation using Lambda LIZ Alpha. The predicted amount of plaques were observed when the host cell was wild-type SCS-15 (supF) lacking plasmid pMI/S 5 . In two individual clones of transformed host SCS-
  • the pMI/S 5 plasmid produced sufficient S 5 protein to block plaque formation.
  • At dosages of 250 plated phage particles no plaques were observed, and at dosages of 2.5 x 10 6 plated phage particles, 7-10 plaques were observed, producing a leakiness frequency of about 1 plaque per 10 5 phage particles.
  • This amount of lysis is acceptable for the assay if color screening is also used because the plaques that do form will be clear rather than blue because only plaques formed in the presence of lad mutations will appear blue; in the presence of wild type lad, lacZ alpha on Lambda LIZ Alpha will be repressed and no beta-galactosidase will be formed to yield the blue color.
  • the S 5 system provides an example of a selectable system in which mutation in the test gene can be measured by two modes of reporter gene phenotype, simultaneously, namely by a selective growth advantage, and by the presence of a color selection.
  • the test gene product laql
  • the test gene product controls both a first reporter gene transcription unit, namely the S 5 reporter gene, and controls a second gene (lacZ alpha) that can also function as a second reporter gene.
  • the second reporter gene allows for the capability to produce a detectable color indicator in the host cell.
  • the above result indicates that the S 5 mutant is a suitable reporter gene for use in the reporter gene inactivating system described in Figure 11.
  • the completed host vector is prepared by introducing the TacP promoter under the control of a lac operator (lacO) .
  • lacO lac operator
  • a PCR-generated fragment containing TacP-lacO (the "ideal" lac operator sequence described herein) is produced using the plasmid vector pTL21 (Stratagene) and oligonucleotide primers p9 (SEQ ID NO 13) and plO (SEQ ID NO 14) having the sequences as follows: p9 5 '-ATCAGCACGCGTTTAATGTGAGTTAGCTCACTCA-3 ' PlO 5'-AGCATCAGATCTCAGCTTTTGTTCCCTTTAG-3 '
  • the resulting PCR-amplified fragment was digested with Mlul and Bglll, treated as before and ligated into the pMI/S 5 that was pre-digested with Ml
  • the resulting plasmid pMI/S 5 tac-lac upon transformation into E. coli SCS-8, is used in the host cell for assaying a test DNA sequence such as Lambda LIZ Alpha recovered from a mutagenized mouse. Detection of the mutagenized lad gene is indicated by the presence of blue plaques when the host cell is cultured as described earlier in the presence of X- gal.
  • the system described in Figure 11 is a prototype for the use of first and second convergent promoters, where the first promoter (lacO) is regulated by the test DNA gene product (lad) and the second promoter (PR 1 ) is regulated by an anti-terminator protein.
  • Terminor/anti-terminator protein systems can be readily utilized in the groE or S 5 based assay systems described in Examples 9 and 10.
  • suitable terminator nucleotide sequences are well characterized, including the Box-A like consensus sequence C/TGCTCTT(T)A (SEQ ID NO 15), and the related transcription terminator sequences for crp, trp, his. phe. thr, ampC, ilv, and rrnB.
  • terminator/anti- terminator system provided by lambda in the nutR-tRl nucleotide sequence and the N protein.
  • the nutR-tRl nucleotide sequence can readily be substituted into the above described terminator/anti-terminator constructions in place of qut-t6s.
  • a PCR-amplified fragment containing the nutR-tRl sequence [that contains tRl(I-III)] is isolated by conducting PCR on wild-type lambda in the presence of oligonucleotide primers pll (SEQ ID NO 16) and pl2 (SEQ ID NO 17) having the sequences: pll 5'-ATCAGCCTGCAGGTCGACTAAATAAACCCGCTCTTACAC-3' P12 5'-AGCATCGTCGACGGATCCCACGAACCATATGTAAGTATT-3 '
  • the resulting PCR fragment is cloned into the above host vector plasmids as described above.
  • the anti-terminator protein N is provided in trans by the lambda test DNA sequence Lambda LIZ Alpha.
  • the host reporter gene constructs can also be present as lower copy number, or as single copy genomic elements within the E. coli chromosome.
  • the preparation of stably- integrated genetic elements was described earlier using the suicide vector system for the construction of the prolysogen strain of E. coli. That system can readily be utilized to produce stably integrated groE or S 5 reporter gene constructions as described herein.
  • a plasmid vector containing a reporter gene under the transcriptional control of a lac promoter and lac operator is constructed having the E. coli groE as the reporter gene and the lambda anti-terminator nucleotide sequence nutR, that is regulated by lambda terminator protein N, and one or more of either of the lambda terminator sequences tRl or t6s, or both.
  • a strain of E. coli that is GroE " is required as the host cell for use of this reporter gene construct, and was prepared earlier in Example 9A, designated SCS-8 groES30, and is tet r .
  • the reporter gene plasmid construct used in a GroE system with the N anti-terminator protein can take a number of various forms, which can vary in the precise reporter gene, including groES, groEL or groESL, and can vary in the structure of the terminator(s) .
  • Plasmid pALS74 carries a nutR site for anti- termination, and has two primary terminators.
  • the first terminator is the lambda terminator tRl that has been shown to consist of a series of five terminator sequences, designated tRl(I-V), to connote the presence of all five sequences.
  • the second terminator is the lambda t6s terminator.
  • pALS74 utilizes the groES reporter gene.
  • Plasmid pALS74 was produced by cloning the lambda nutR-tRl(I-V) region by PCR into plasmid pl5G10Qut described earlier in Example 9 to form plasmid pl5G10Nut, and then moving the fragment that contains the elements lacP-lacO-nutR-tRl(I-V) -t6s-groES into the Bglll site of the low copy number vector pALlow to form pALS74.
  • Plasmid pALS74 was then tranformed into the E. coli host SCS-8 groES30 described in Example 9 to form SCS-8 groES30(pALS74) .
  • the plasmid pALS74 has been deposited with the ATCC in the form of E. coli strain SCS-8 groES30 described herein and containing the plasmid pALS74.
  • Plasmid pALS74 is a low copy number plasmid because it contains the RSF1010 bacterial origin of replication.
  • the reporter construct on plasmid pBQ is very similar to pALS74 and can be represented by the following schematic: ... -lacP-lacO-nutR-tRl(I-V) -t6s-t6s-groESL....
  • Plasmid pBQ carries a nutR site for anti-termination, and has three primary terminators.
  • the first terminator is the lambda terminator tRl(I-V), and the second and third terminators are each the lambda t6s terminator.
  • pBQ utilizes the groESL reporter gene.
  • Plasmid pALS74 is restriction endonuclease digested with Apal and Sail to release a fragment containing the groES gene. The resulting fragments are treated with CIAP and the larger
  • vector fragment is gel purified to form a vector fragment.
  • Plasmid pBSG3 was prepared as described in Example 9, and is restriction endonuclease digested with Apal and Sail to release a 2.2 kilobase (Kb) fragment containing the groESL gene.
  • the groESL fragment is similarly gel purified and ligated with the above vector fragment to form pALS74 (SL) .
  • pALS74(SL) is then restriction endonuclease digested with. EcoRI and Sail, treated with CIAP, and the largest (vector) fragment is gel purified to form phosphatased pALS74 (SL) linear vector.
  • Wild-type lambda is then PCR-amplified using Pfu DNA polymerase (Stratagene) under standard PCR conditions with the following primers pl3 (SEQ ID NO ) and pl4 (SEQ ID NO ) : pl3 5 ' -CGGATCGAATTCATGGGTTAATTCGCTCGTTGT-3 ' pl4 5 '-GCGAGTGTCGACTCTTACCTGTTGTGCAGATAT-3 '
  • the resulting PCR-amplified fragment of about 215 bp contains the lambda t6s terminator sequence. Thereafter, the fragment is restriction endonuclease digested with EcoRI and Sail, gel purified and ligated with the phosphatased pALS74(SL) linear vector to form pBQ.
  • the reporter construct on plasmid pBQ2 is very similar to pBQ, and can be represented by the following schematic:
  • Plasmid pBQ2 carries a nutR site for anti-termination, and has three primary terminators. The first terminator is the lambda terminator tRl(I-II), and the second and third terminators are each the lambda t6s terminator. In addition, pBQ2 utilizes the groESL reporter gene.
  • pBQ is restriction endonuclease digested with NotI and Spel to release a fragment of about 270 bp containing the nutR-tRl(I-V) genes.
  • the resulting fragments are treated with CIAP and the larger (vector) fragment is gel purified to form phosphatased linear pBQ vector fragment.
  • Wild-type lambda is then PCR-amplified using Pfu DNA polymerase under standard PCR conditions with the following primers pl5 (SEQ ID NO ) : 5 • -ATCGACAGCGAATTCGCGGCCGCATAAATAACCCCGCTCTTACAC-3 * and pl6 (SEQ ID NO ) :
  • the resulting PCR-amplified fragment of about 150 bp contains the nutR-tRl(I-II) region of lambda.
  • the fragment is restriction endonuclease digested with NotI and Spel, gel purified and ligated with the phosphatased linear pBQ vector fragment to form pBQ2.
  • the reporter construct on plasmid pBQ2C is very similar to pBQ, and can be represented by the following schematic: ... -lacP-lacO-nutR-tRl(I-II)-t6s-t6s-T7rbs-groES....
  • Plasmid pBQ2C carries a nutR site for anti- termination, and has three primary terminators, and a T7 gene 10 ribosome binding site (T7rbs) .
  • the first terminator is the lambda terminator tRl(I-II)
  • the second and third terminators are each the lambda t6s - terminator.
  • pBQ2C utilizes the groES reporter gene.
  • pBQ2 is restriction endonuclease digested with Sail and Apal to release a fragment containing the groESL gene.
  • the resulting fragments are treated with CIAP and the larger (vector) fragment is gel purified to form phosphatased linear pBQ2 vector fragment.
  • Plasmid pBSG3 produced in Example 9 is then PCR- amplified using Pfu DNA polymerase under standard PCR conditions with the following primers pl7 (SEQ ID NO
  • pl8 SEQ ID NO : 5 '-CCTCTCGAGGGGCCCCTATTACGCTTCAACAATTGCCA-3 '
  • the resulting PCR-amplified fragment of about 300 bp contains the groES gene, and has a T7 genelO ribosome binding site (rbs) in place of the groES rbs based on the design of the PCR primer. Thereafter, the fragment is restriction endonuclease digested with Apal and Sail, gel purified and ligated with the phosphatased linear pBQ2 vector fragment to form PBQ2C.
  • rbs T7 genelO ribosome binding site
  • Example 9 selection is carried out essentially as described in Example 9 by transforming an E. coli host cell that is GroE " , such as SCS-8 groES30. and screening a target DNA sequence such as Lambda LIZ Alpha which expresses the anti-terminator protein N. Phage plaque formation is inhibited in a manner analogous to the mechanism shown in Figure 10 because groE gene products are required for phage assembly and the transcription of the groE reporter gene transcription unit is inhibited by the terminator sequences until the lambda infects and produces the lambda N anti-terminator protein. Expression of wild type lacl from Lambda LIZ Alpha then inhibits reporter gene transcription. Transcription will occur if mutant lad (non-repressing) is present.
  • Low copy vectors using the groE selection system are preferred because they exhibit greater sensitivity than the medium copy vectors.
  • All of the repoerter vector constructs described in Example 11 are low copy vectors having the RSF1010 origin of replication.
  • the selection systems based on GroE, Sy5 or lactose dependent growth in prolysogenic host cells described in Examples 11, 10 and 8, respectively, have been compared for efficiency and sensitivity. The selection systems allow for significantly more rapid analysis because larger numbers of packaged phage particles can be screened per assay plate.
  • phage particles are plated per plate when using color detection in a non- selectable system based on lad as the target gene as described in Example 4D(2)
  • 100,000 phage particles are plated per plate when using GroE or approximately 45,000 particles per plate for SY5 selection
  • about 200,000 phage particles are plated per plate when using lactose selection with a prolysogenic host. Mutagenesis testing was also compared using the various systems described herein.
  • Plaques or colonies measured are expressed as xlO "5 .
  • the systems used are (1) the non-selectable color based assay, (2) lactose selection using prolysogen SCS-8cI, (3) S5 selection described in Example 10, and (4) GroE selection using pALS74.
  • Table 3 illustrate that the various selection systems described herein allow the detection of mutagenic activity in transgenic animals.

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Abstract

L'invention décrit un procédé d'analyse pour contrôler et évaluer le potentiel de mutagénèse d'agents, ce procédé consistant à créer et à utiliser des organismes non humains transgéniques portant une ou des séquences d'ADN test qui peuvent être examinées du point de vue de leurs mutations spontanées ou de mutations induites, après exposition à un ou plusieurs agents mutagènes présumés. Dans un système d'analyse préféré, les séquences d'ADN test ayant subi la mutagénèse peuvent être sélectionnées, ce qui augmente la sensibilité de l'analyse.
EP93906965A 1992-02-14 1993-02-12 Test de mutagenese utilisant des animaux non humains transgeniques portant des sequences d'adn test. Withdrawn EP0671955A4 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US83703192A 1992-02-14 1992-02-14
US837031 1992-02-14
US94119092A 1992-09-04 1992-09-04
US941190 1992-09-04
PCT/US1993/001293 WO1993015769A1 (fr) 1992-02-14 1993-02-12 Test de mutagenese utilisant des animaux non humains transgeniques portant des sequences d'adn test

Publications (2)

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EP0671955A1 EP0671955A1 (fr) 1995-09-20
EP0671955A4 true EP0671955A4 (fr) 1997-04-09

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EP93906965A Withdrawn EP0671955A4 (fr) 1992-02-14 1993-02-12 Test de mutagenese utilisant des animaux non humains transgeniques portant des sequences d'adn test.

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EP (1) EP0671955A4 (fr)
CA (1) CA2130081A1 (fr)
WO (1) WO1993015769A1 (fr)

Families Citing this family (8)

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Publication number Priority date Publication date Assignee Title
GB9616685D0 (en) 1995-10-02 1996-09-25 Cancer Res Campaign Tech Antitumour vector constructs and methods
CA2319114A1 (fr) * 1998-01-30 1999-08-05 Donald L. Heefner Proteines de fusion regulatrices de genes et leurs procedes d'utilisation pour determiner la resistance d'une proteine a un medicament dirige contre elle
US6307121B1 (en) 1998-05-31 2001-10-23 The University Of Georgia Research Foundation, Inc. Bacteriophage-based transgenic fish for mutation detection
AU1323000A (en) 1998-10-26 2000-05-15 University Of Georgia Research Foundation, Inc., The Transgenic fish carrying plasmid for mutation detection and methods
US6653075B2 (en) 1999-12-16 2003-11-25 Iconix Pharmaceuticals, Inc. Random domain mapping
EP1176211A1 (fr) * 2000-07-27 2002-01-30 Novartis Forschungsstiftung, c/o Novartis International AG Procédé de surveillance des mutagenes dans les plantes
GB0602173D0 (en) 2006-02-03 2006-03-15 Avecia Ltd Expression system
WO2009073551A2 (fr) * 2007-11-30 2009-06-11 Scarab Genomics Llc Système d'expression de lac

Citations (4)

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Publication number Priority date Publication date Assignee Title
WO1989009272A1 (fr) * 1988-03-22 1989-10-05 Chemical Industry Institute Of Toxicology Souris transgenique pour la mesure et la caracterisation de mutations induites in vivo
EP0370813A2 (fr) * 1988-11-25 1990-05-30 Exemplar Corporation Dosage rapide par criblage de mutagénèse et tératogénèse
WO1991015579A1 (fr) * 1990-04-05 1991-10-17 Stratagene Test de mutagenese a l'aide d'animaux transgeniques porteurs de sequences d'adn tests
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 (3)

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Publication number Priority date Publication date Assignee Title
US4736866A (en) * 1984-06-22 1988-04-12 President And Fellows Of Harvard College Transgenic non-human mammals
EP0289121B1 (fr) * 1987-05-01 1995-12-27 Stratagene Test de mutagénèse utilisant des êtres non humains transgéniques porteurs des séquences d'ADN à essayer
NL8801826A (nl) * 1988-07-19 1990-02-16 Tno Werkwijze voor het detecteren van mutaties in markergenen.

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989009272A1 (fr) * 1988-03-22 1989-10-05 Chemical Industry Institute Of Toxicology Souris transgenique pour la mesure et la caracterisation de mutations induites in vivo
EP0370813A2 (fr) * 1988-11-25 1990-05-30 Exemplar Corporation Dosage rapide par criblage de mutagénèse et tératogénèse
WO1991015579A1 (fr) * 1990-04-05 1991-10-17 Stratagene Test de mutagenese a l'aide d'animaux transgeniques porteurs de sequences d'adn tests
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 (3)

* Cited by examiner, † Cited by third party
Title
J.S. LEBKOWSKI ET AL.: "The lacI shuttle: Rapid analysis of the mutagenic specificity of ultraviolet light in human cells", PROC. NATL.ACAD SCI., vol. 82, December 1985 (1985-12-01), NATL. ACAD SCI.,WASHINGTON,DC,US;, pages 8606 - 8610, XP002023904 *
P.M. GLAZER ET AL.: "Detection and analysis of UV-induced mutations in mammalian cell DNA using a lambda shuttle vector", PROC. NATL.ACAD SCI., vol. 83, February 1986 (1986-02-01), NATL. ACAD SCI.,WASHINGTON,DC,US;, pages 1041 - 1044, XP002023905 *
See also references of WO9315769A1 *

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EP0671955A1 (fr) 1995-09-20
CA2130081A1 (fr) 1993-08-19
WO1993015769A1 (fr) 1993-08-19

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