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WO1993017123A1 - Test de la mutagenicite a l'aide de genes rapporteurs avec frequences de methylation modifiees - Google Patents

Test de la mutagenicite a l'aide de genes rapporteurs avec frequences de methylation modifiees Download PDF

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Publication number
WO1993017123A1
WO1993017123A1 PCT/US1993/001676 US9301676W WO9317123A1 WO 1993017123 A1 WO1993017123 A1 WO 1993017123A1 US 9301676 W US9301676 W US 9301676W WO 9317123 A1 WO9317123 A1 WO 9317123A1
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gene
cpg
marker
ala
animal
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PCT/US1993/001676
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English (en)
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Peter J. Stambrook
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Ohio University
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • 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
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/108—Swine
    • 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
    • A01K2227/40—Fish
    • 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

Definitions

  • This invention relates to testing for mutagens and carcinogens.
  • Carcinogens are chemical (or physical) agents which are capable of causing cancer in a susceptible subject.
  • a chemical is considered a carcinogen if, in a well designed and conducted bioassay, it produces a statistically significant increase in the incidence of neoplasms in one or more target organs.
  • Some carcinogens are themselves mutagens, i.e., agents which directly cause the mutation of DNA. Others are metabolized by cells to form powerful mutagens, which in turn act on the host's DNA.
  • aflatoxin Bl is converted by a hepatic aryl hydroxylase into 2,3-epoxide derivative, a mutagen, and nitrates are a starting material in the formation of the highly carcinogenic (mutagenic) nitrosamines.
  • Still others act as tumor initiators or promoters, i.e., they induce or accelerate the transformation of normal cells into maliganant cells without necessarily directly modifying the genetic material.
  • carcinogens and mutagens were identified by epidemiological means. After several decades, segments of the population having elevated exposures to the agent would exhibit a higher frequency of incidence of a particular cancer or other disorder. Case studies would reveal the common factor. Of course, the problem with epidemiological detection is that it is retrospective; society has already been damaged. This prompted the development of a variety of screening tests.
  • the Ames test is based on the assumption that carcinogens (or their metabolites) will cause the genetic reversion of certain mutant strains of bacteria. These strains lack the ability to prc-duce histidine, an essential amino acid, and therefore are unable to multiply unless this nutrient is in their growth medium. In the presence of a mutagen, these mutants are more likely to revert to their "wild" phenotype, i.e., they regain the ability to manufacture histidine from other materials and therefore can grow in a histidine-free medium.
  • a chemical may be converted into a carcinogen or mutagen through a metabolic activity specific to a particular type of cell, e.g., periportal liver cells. Unless, fortuitously, cells of this type are exposed to the chemical, this mode of carcinogenesis or mutagenesis will not be discovered.
  • the production of the mutagen requires processing by several different types of cells, or that the necessary metabolic activity in one type of cell must be activated by a product of a different type of cell.
  • a single cell type bioassay is incompetent for risk assessment of chemicals which are metabolized in this manner.
  • transgenic animals were prepared whose cells carried a foreign (typically bacterial) "marker" gene. These animals were exposed to normal doses.of the suspect chemical. DNA was then extracted from the various tissues and organs of the animal, and the foreign "marker” gene was "rescued” and transferred to the genome of a host by means of a bacteriophage. The lytic plaques were then screened for the phenotype characteristically imparted by the original "marker” gene. The absence of this phenotype was indicative of mutation.
  • a phage lambda vector capable of lysing E. coli, was engineered to carry the E. coli beta galactosidase (lacZ) gene.
  • Lambda DNA was microinjected into mouse embryos, and transgenic mice were produced by standard techniques. Genomic DNA was purified from a tissue of the transgenic mouse, and the test DNA was excised by means of a lambda phage packaging extract. The packaged phage were incubated with beta-galactosidase deficient E. coli. Bacteria infected by the phage particles were lysed, resulting in the formation of lytic plaques on a lawn of beta- galactosidase deficient E. coli.
  • the transgenic animal-based test unlike an in vitro mammalian cell bioassay, can detect mutagenesis by metabolites of the chemical of interest, even though the metabolites are produced at the appropriate concentrations only by differentiated cells or the tissue of live animals.
  • Cells of any tissue or organ of interest may be screened for mutagenic damage, merely by extracting their DNA and recovering and characterizing the transgene.
  • a single animal may yield a multitude of cells for testing and analysis. .It should be noted, to avoid confusion, that this cellular level analysis cannot be performed with geries endogenous to the test animal. They cannot be isolated from the DNA of an organ or tissue with sufficient efficiency. That is why this more sophisticated analytical approach was not possible until it became feasible to make transgenic animals.
  • the present invention overcomes the deficiencies of the test methods described above.
  • the present Applicant recognized that bacterial genes exhibit a much higher frequency of occurrence of the "CpG" doublet than do vertebrate genes. As a result, a bacterial gene incorporated into a mammalian genome will exhibit a much higher degree of methylation than is typical for a mammalian gene.
  • the primary nucleic acid methylation substrate in mammals For example, of the 1081 dinucleotides in the lad gene, 95 (about 9%) are CpG, the primary nucleic acid methylation substrate in mammals. (55 of these CpG lie within a single one of the 360 codons of the gene, and 40 are at intercodon boundaries.)
  • the mammalian gene is underrepx * esented in CpG dinucleotides. In random DNA having a 50% GC content, about 6% of the dinucleotides would be expected to be CpG. In mammalian DNA, it occurs with a frequency of about 2%. .
  • This problem may be overcome by the use of a wholly or partially synthetic bacterial marker gene having a reduced (vertebrate cell like) number of CpGs, and hence, presumably, a lower overall level of methylation.
  • Vijg had been troubled by the very low number of plaques obtained in practice. He postulated that the methylation pattern of the lambda vectors was rendering them susceptible to restriction by the E. coli host, i.e., the lambda vectors were being cut to pieces by the bacterium's defensive enzymes before they could integrate into the host genome (Vijg, page 3) . His solution, however, was not to modify the vector, but rather to employ a "host restriction"- negative strain for plating.
  • Figure 1 sets forth the sequence of the coding strand of the wild-type lad gene, 5" to 3' (SEQ ID N0:1) . CpG dinucleotides are marked. Above the nucleotide sequence are alternative nucleotides for eliminating most of the CpG dinucleotides and for eliminating splicing donor-acceptor sites. Below the nucleotide sequence is the sequence of the corresponding wild- type Lad repressor protein (SEQ ID N0:2) , given according to the single letter amino acid code.
  • Figure 2 sets forth the sequence (LACIMIRNL) (SEQ ID NO:3) of a Kozak consensus RBS (TCACC.,..), a CpG-depleted lad gene, a three codon linker (encoding AAL) , and a seven codon sequence encoding the SV40 large antigen nuclear localization site. These features are marked, as are all Mspl/Hpall (CCGG) sites.
  • the transgene also includes a beta actin promoter, but, since this sequence is lengthy and has been published, it was not reprinted. The corresponding amino acid sequence is presented as SEQ ID N0:4.
  • Figure 3 shows the wild-type E. coli gpt gene (SEQ ID NO:5) and the suggested base substitutions for reducing its CpG content.
  • the corresponding amino acid sequence is presented as SEQ ID NO:6.
  • Figure 4 shows the modified gpt gene (SEQ ID NO:7) and a synthesis strategy therefor.
  • the complementary DNA sequence is provided as SEQ IS NO:8.
  • Figure 5 is a schematic depiction of (A) a Hindlll/BamHI fragment comprising the lacZ gene and SV40 processing signals, and (B) plasmid pL26.6.
  • Figure 6 shows the construction of plasmid PAP lacOZneo.
  • Figure 7 depicts the organization of the mouse aprt promoter region with the site of one lacO insert marked.
  • the aprt promoter and upstream sequences that will be used in the proposed experiments are schematically displayed. In the genome, this fragment is bounded by Fnudll sites. The numbering in base pairs begins at the translation start codon.
  • the 4 boxes represent sites of Spl binding.
  • the horizontal arrows indicate major sites of transcription initiation, and the vertical arrow indicates extend of deletion that permits full aprt expression.
  • the position of the E. coli lac operator (lacO) is indicated, as are potential Taql and Xmal sites for alternative operator insertion.
  • the present invention is directed to a test for mutagenicity and carcinogenicity, in which mutations in a marker gene are used to predict the effect of a chemical agent on genes of a target species of interest.
  • the target species will be human beings, however, it must be noted that both wild and domesticated animals are also chronically exposed to potential mutagens and carcinogens and that environmental policy may call for minimizing such exposure.
  • the present invention may readily be adapted to screening for the mutagenic potential of a chemical vis-a-vis the genes of a nonhuman animal species, including other mammals, birds, fish, amphibia, reptiles, and even lower life forms (such as bees, silkworms, earthworms, and so forth) .
  • Nucleic acids are the basic genetic material in cells. They are formed by a chemically linked sequence of nucleotides. Each nucleotide contains a heterocyclic ring of carbon and nitrogen atoms (the nitrogenous base) , a five carbon sugar in ring form (a pentose) , and a phosphate group. Two types of pentoses are found in nucleic acids, the 2-deoxyribose in DNA (deoxyribonucleic * acid) and the ribose in RNA (ribonucleic acid) .
  • RNA In DNA, there are four normal nitrogenous bases: two pyrimidines, cytosine (C) and thymine (T) , and two purines, adenine (A) and guanine (G) .
  • C cytosine
  • T thymine
  • U uracil
  • a base-sugar moiety is called a nucleoside
  • a base-sugar-phosphate moiety is a nucleotide. Genetic information is conveyed by the sequence of bases in a polynucleotide chain, not by the phosphodiester-sugar backbone.
  • the 5' position of one pentose ring is connected to the 3' position of the next pentose ring via a phosphate group, thus forming a series of 5' to 3' linkages.
  • the terminal nucleotide at one end of the chain has a free 5' group, and the other terminal nucleotide has free 3' group.
  • IT is conventional to write nucleic acid sequences by setting forth the sequence of bases in the 5' to 3' direction.
  • the chromosome is a double helix formed by two very long, interweaving polynucleotide chains, or strands. These strands are held together in the double helical structure as a result of hydrogen bonds between so-called complementary bases on the two strands.
  • One such "base pair”, adenine-thymine (A:T) (or, in RNA, adenine-utacil, A:U) provides two hydrogen bonds; the other, guanine-cytosine (G:C) . provides three.
  • RNA transcript When a gene in the chromosome is expressed, a single strand of messenger RNA is transcribed from a template strand of the chromosome by complementary base pairing.
  • the template strand is called the coding or anti-sense strand; the other DNA strand, the anti-coding or sense strand, is identical in sequence (except for the T/U distinction) to the messenger RNA transcript.
  • the messenger RNA acts as a template for the assembly of amino acids into the protein encoded by the gene; the assembly process is known as translation.
  • the messenger RNA transcript is read in nonoverlapping units of three nucleotides, known as codons, from a fixed starting point; there are three possible ways of translating any messenger RNA, depending on the starting point. These are known as reading frames.
  • the bases of DNA may be modified by enzymes endogenous to the cell, especially methylases. In vertebrates, the only methylated base is 5-methylcytosine. Between 2% and 7% of the C residues of animal cell DNA are methylated. Most of the methyl groups are found in CpG "doublets" (dinucleotides) ; in birds and mammals, 50- 70% of all such dinucleotides are modified by methylation.
  • the C and G are adjacent bases on the same strand, joined by a covalent 5 1 to 3' phosphodiester(p) -sugar linkage; the CpG dinucleotide should not be confused with the C:G base pair formed by hydrogen bonding between a C on one strand and a G on another.
  • a doublet that is instead methylated on only one of the two strands is said to be hemimethylated.
  • the distribution of methyl groups may be examined by taking advantage of pairs of restriction enzymes, known as isoschizomers, that cleave the same target sequence in DNA, but have a different sensitivity to its methylation pattern.
  • the enzymes Hpall and Mspl both recognize the sequence CCGG, which includes a CG dinucleotide.
  • CCGG which includes a CG dinucleotide.
  • Mspl is indifferent to the presence of methylation at this C.
  • Mspl can be used to identify all the CCGG sites, and Hpall, to determine whether or not they are methylated.
  • some methylation sites are methylated when the gene is inactive but unmethylated (at least in some cells) when the gene is expressed.
  • Palmiter and Brinster Ann. Rev. Genet., 20:465- 99 (1986)
  • the methylation of DNA microinjected into mouse embryos, in the course of transgenic mouse production may in some cases be responsible for problems observed in expressing transgenes.
  • a mutation is a change in the nucleotide sequence. An alteration that alters only a single base pair is termed a point mutation.
  • a point mutation may take the form of a substitution, an insertion, or a deletion.
  • a point mutation in a gene will not necessarily have an effect on the sequence of the encoded polypeptide. This is because the genetic code is redundant--61 different codons encode only twenty different amino acids. -A mutation which does not affect which amino acid is encoded is termed a "silent" mutation. Even if the amino acid is changed, it is possible that the mutant polypeptide will retain activity. In this case the mutation is said to be "neutral".
  • a mutation which inactivates a gene is termed a forward mutation.
  • the effects of forward mutations may be reversed by back mutations in the same genes, or through suppression of the mutated gene through mutation of a different gene.
  • Back mutations fall into two categories, true reversions, which restore the wild- type sequence, and second- site reversions, which simply compensate for the forward mutation by restoring activity.
  • a nucleotide in a gene will cause a shift of the reading frame. In general, this will result in the expression of a radically different and probably nonfunctional polypeptide. However, a second frameshift mutation, close enough to the first one, may restore activity.
  • Mutations may also involve the insertion, deletion, or inversion of larger chunks of DNA.
  • Nitrous acid deaminates adenine to hypoxanthine, whichbonds to cytosine instead of to thymine.
  • the new strand features a cytosine, rather than a thymine, at the position complementary to the hypoxanthine.
  • the polymerase inserts a guanine, which is complementary to the aforementioned cytosine.
  • nitrous acid causes an A->G transition. Cytosine is deaminated to uracil, which hydrogen bonds to adenine instead of to guanine.
  • nitrous acid also causes a C->T transition.
  • Guanine is deaminated to xanthine, which continues to hydrogen bond to cystosine, though with only two hydrogen bonds.
  • Thymine and uracil are not altered by nitrous acid.
  • Hydroxylamine reacts with cytosine to form N- hydrocytosine, which preferentially pairs with adenine.
  • hydroxylamine produces a C->T transition.
  • the alkylating agents are the largest group of mutagens, which introduce alkyl groups into nucleotides at various positions.
  • the alkylating agents include mustard gas, epoxides, dimethyl- and diethylsulfonate, methyl- and ethylmethane- sulfonate, and N-methyl-N" -nitroso-N-nitroguanidine.
  • G->A (as a result of formation of 0 6 -alkylguanine)
  • T->C attributable to O 6 -alkylthymine
  • the present invention is not limited to the evaluation of any particular chemical class of potential mutagen, or to the detection of any particular kind of mutation.
  • methylated cytosines also acts as a hotspot for mutation in the genes of vertebrate cells, it follows that the placement of a heavily methylated marker gene in a vertebrate cell for mutagenesis assay purposes will result in overestimation of the mutagenic potential of the assayed chemical.
  • transgenic animal is defined, for the purpose of the appended claims, as an animal at least some of whose germ cells contain genetic material, originally derived from another animal, other than an ancestor of said animal, as a result of human intervention. So defined, it includes progeny of a transgenic animal which retain the transgenic genotype. It is not necessary that all cells of the animal contain the transgene.
  • the reference to human intervention is intended to exclude genetic modification as a result of unintentional infection with a virus.
  • chimeric animal is defined, for the purpose of the appended claims, as an animal which is not necessarily a transgenic animal, but at least some of whose somatic cells contain genetic information, originally derived from another animal other than an ancestor of said animal, as a result of human intervention.
  • genetic engineered animal refers to an animal which is either a transgenic animal or a chimeric animal.
  • animals produced by conventional artificial insemination techniques are not considered to be genetically engineered, the donors of sperm and egg being considered parents of the animal, unless one or more ancestors of the animal was genetically engineered and the descendant animal retains the engineered genotype.
  • the transplantation of cells from one animal to another is not considered genetic engineering.
  • transgenic animals may utilize both transgenic animals and chimeric animals, though transgenic animals are preferred. References to transgenic animals in this specification should be deemed to include, mutatis mutandis. chimeric animals as well.
  • the "marker gene” may be any gene which confers a selectable or screenable phenotype on cells of the transgenic animal, or, if the assay is not applied directly to those cells, on the assay cells subsequently transformed by the rescued marker gene.
  • the marker gene is . one which is not substantially homologous with any gene endogenous to the host animal from which the transgenic animal is produced. This facilitates the identification of the marker gene.
  • the marker gene will be a bacterial gene in order to maximize the taxonomic distance between the marker gene and the genes of the host animal. However, it may also be a nonbacterial, nonmammalian gene, uch as a viral, fungal, plant, invertebrate or lower vertebrate gene.
  • the marker gene may be a wild type nonvertebrate gene chosen because it has a CpG level in the vertebrate range; more often, it is a nonvertebrate gene mutated to reduce the CpG level.
  • the detected mutation in the marker gene may be any of the numerous types of mutation known to occur. It may be in the coding sequence of the DNA, or in an associated regulatory sequence such as a promoter or a stop codon. It may be a point mutation or a frameshift mutation. It may involve a base substitution, insertion or deletion.
  • the marker gene is a functional gene, and the assay is for forward mutations which inactivate the gene.
  • the marker gene is one mutated to render it nonfunctional, and the assay is for back mutations which restore activity.
  • the second of these embodiments has the advantage .of lower background, since back mutation is a rare event.
  • the marker gene may be a lacZ gene with the codon for Glu- 461 (GAA) mutated to HBA; it has been shown in IjL. coli that only same site reversion will restore activity.
  • the phenotypic change (marker) associated with mutation of the marker gene may be one detectable in a mammalian cell, or it may be one detectable only after rescue of the mutated DNA and expression of that DNA in a non- mammalian, e.g., bacterial system.
  • the mutagenesis assay may detect a direct or an indirect product of the marker gene.
  • the detection may occur in vivo or in vitro, through any means known in the diagnostic art.
  • the phenotypic change may be the death of the animal or the affected cells, or a change in cell morphology or metabolism. It may be the presence or absence of a characteristic luminescence or radioactivity. All that matters is that there be a detectable change if mutation occurs. It is not necessary that all mutations cause a detectable change, as long as some mutations in the marker gene will do so.
  • the detection could be through in vitro examination of the blood, urine, milk, or other expendible product of the animal. This has the advantage that the animal is not harmed, so that the animal can continue to be monitored for further genotoxic damage from the agent. However, there is no way of knowing how the detected label enters the examined product, and hence it is not known whether the mutation occurs only in certain tissues or organs.
  • the detection could be through histochemical examination of one or more tissues of the animal. See, e.g., Wei, EP Appl 370,813.
  • the marker gene may be, but is not limited to, a tumorigenic, toxin, hormonal, enzymatic or antigenic marker gene. (It should be noted that these categories are not mutually exclusive.)
  • Tumorigenic marker genes are those which, when expressed in a transgenic animal, result in production of a transforming gene product and therefore induce tumors.
  • the marker gene may be a functional oncogene, in which case the assay is for mutations which render the oncogene nonfunctional and therefore protect the animal, or it may be an oncogene mutated to be nonfunctional, in which case the assay is for back mutations which render the oncogene functional once more and therefore result in tumor formation in the animal.
  • the oncogene may be a viral or a cellular oncogene.
  • the marker gene may be a naturally occurring proto-oncogene, or it may be an oncogene mutated in the laboratory to render it nonfunctional.
  • the oncogene When the oncogene is a viral oncogene, it may be derived from a DNA tumor virus or from a retrovirus.
  • Suitable retroviral oncogenes include v- abl, v-fes, v-fps, v-fgr, v-src, v-erbA, v-erbB, v-fms, v-ros, v-yes, v-mos, v-ras, v-fos, v-myb, v-myc, v-ski, v-sis, v-rel, v-kit, v-jun, andv-ets.
  • Suitable DNA tumor virus genes include the T antigen genes from SV40 or polyoma viruses and the EIA and E1B genes from adenoviruses.
  • Toxin marker genes encode a toxic protein or an enzyme which participates in the enzymatic production of a toxic metabolite.
  • the toxin may be, but is not limited to, a bacterial toxin (e.g., diphtheria toxin, tetanus toxin, and botulin toxin) , a plant toxin (e.g., ricin or abrin) , an invertebrate toxin (e.g., a scorpion or sea anemone toxin), or a snake venom toxin (e.g., a cobra or rattlesnake toxin) .
  • Toxins include cardiotoxins, neurotoxins, and protease inhibitors. Nonfunctional mutants of toxin genes may be used in back mutation assays.
  • Hormonal marker genes encode protein or peptide hormones (or prohormones, or pre-prohormones) which are detectable either directly or through their biological effect. These hormones may be identical to natural counterparts secreted by, e.g., the endocrine glands (such as the pituitary, thyroid, or gonads) , or they may be muteins. Suitable hormones include growth hormone, prolactin, chorionic gonadotropin, luteinizing hormone, follicle stimulating hormone, insulin, parathyroid hormone, somatostatin, and gonadotropin releasing hormone, and homologues thereof. While most mammalian hormonal marker genes will exhibit CpG frequencies typical of mammalian DNA, exceptions may exist. Also, nonmammalian hormonal marker genes may be of interest as their proteins may be more readily differentiated from their mammalian cognates in a transgenic mammalian host.
  • Enzymatic marker genes encode enzymes which, in the presence of a suitable substrate, convert the substrate into a directly or indirectly detectable product. Suitable enzymes include beta- galactosidase (lacZ) , alkaline phosphatase, luciferase and horseradish peroxidase.
  • Enzymatic marker genes are particularly appropriate where the marker gene is engineered so that the enzyme is secreted into an assayable biological fluid, such as blood.
  • the substrate can then be supplied when the blood is assayed in vitro. They may also be used when the marker is to be detected by histochemical analysis. In any event, the substrate may be provided in vivo or in vitro.
  • Antigenic marker genes encode a detectable antigen. The antigen is then detected with a specific antibody. Antigenic marker genes are particularly suitable for detection of mutagenic activity by in vivo imaging, as the antibody may be labeled with an imageable label such as a radioactive label. Regulatory marker genes are genes which encode regulatory proteins. Such proteins control the expression of other genes.
  • Examples include the lad repressor gene and the lambda repressor gene, and the lad activator protein LAP267, see Bairn, et al.. PNAS (USA), 88:5072-5076 (June 1991). With regard to lac repressor, see Wyborski and Short, Nucleic Acids Res., 19:17 (Sep. 1991) .
  • Suppressor tRNA Genes encode tRNAs which suppress the effect 5 of a chain termination mutation.
  • the supF gene suppresses the amber mutation and the supE gene the ochre mutation. If there is, for example, an amber mutation in a required or selectable function, the mutation can be suppressed by a functional supF gene. Thus, if there is an amber mutation in a
  • Antibiotic resistance genes include the ampicillin, choramphenicol, neomycin, bleomycin, puromycin resistance genes. The rescue approach is preferred when the marker is an antibiotic
  • the lad and lacZ genes are of particular interest, and it is therefore appropriate to discuss their function in nature.
  • the polycistronic lac operon comprises the lac promoter, the lac operator (lacO) , the lacZ, lacY and lacA genes, and a terminator.
  • lacZYA Immediately 5' of the lac operon is the monocistronic lad operon, which comprises the lad promoter, the lad gene, and a terminator.
  • the lacZ gene encodes the enzyme,beta-galactosidase
  • lacY and lacA encode the enzymes beta-galactoside permease and transacetylase, respectively.
  • 25 gene cluster is normally repressed by the Lad repressor protein, which binds to the lacO operator site and thereby prevents the binding of DNA-directed RNA polymerase to the operator. Transcription is activated if an inducer, such as IPTG, is present; IPTG releases the Lad repressor from the lacO site.
  • an inducer such as IPTG
  • the vector used to introduce the marker gene may contain one copy of a particular marker gene, multiple copies of a single
  • 35 marker gene or several different marker genes. Use of multiple marker genes, whether the same or different, alters the sensitivity of the assay.
  • marker genes of nonvertebrate origin will exhibit a higher frequency of the CpG dinucleotide than do the genes of a vertebrate host animal.
  • the expected CpG frequency in DNA of random sequence is (GC%) 2 /4.
  • the expected CpG frequency is 4%
  • the expected CpG frequency is 9%.
  • Bacteria exhibit CpG frequencies in keeping with statistical predictions. However, for vertebrates, especially mammals, the CpG frequency is depressed overall, though so-called HTF regions are marked by higher-than-expected CpG frequencies. and are usually hypomethylated.
  • the E. coli gpt gene for example, has a CpG frequency of about 8.5%, while for lacl, it is about 9%.
  • this invention may be applied to any marker gene, it is especially suitable for marker genes where the wild-type gene has a CpG frequency substantially higher than is typical of genes of the target species, e.g., at least about twice the frequency (thus, >4% for mammalian target species) , and more preferably at least about four times the frequency (>8% for mammalian target species) .
  • the marker gene in which the CpG dinucleotide frequency is reduced, preferably to the point that it is not substantially greater than the CpG dinucleotide frequency in genes of the target species (e.g., not greater than twice, better yet, VA times) .
  • the marker gene preferably is engineered so that its CpG dinucleotide frequency does not substantially exceed the frequency in mammalian genes, which is 2%.
  • the CpG dinucleotide frequency is 1-3%.
  • Each amino acid of a polypeptide is encoded by a DNA triplet, or codon. Since there are four bases (A,T,C,G) in DNA, there are 4 3 possible triplets. Three -- the stop codons -- direct the termination of the polypeptide chain. The remaining 61 possible codons encode the twenty protogenic amino acids. Each amino acid is encoded by one (Met, Trp) to six (Arg, Ser, Leu) different codons.
  • a CpG dinucleotide pair may be formed by the first and second bases of a codon, as in the Arg codon CGT. by the second and third bases of a codon, as in the Thr codon ACG. or by the last base of one codon and the first base of the next one, as in the Cys-Ala encoding sequence TGC.GCA. The last situation is called an "intercodon CpG".
  • the Met (ATG) , Trp (TGG) , Lys (AAA, AAG) and Gin (CAA, CAG) codons are incapable of forming a CpG dinucleotide.
  • Table A sets forth five amino acids for which there is at least one CpG-containing codon, and lists the alternative codons, with the percentage of usage of that codon of all codons encoding the same amino acid, in mammals, give in parenthesis. (Codon preferences for non-mammalian vertebrates are also available.)
  • the next table (B) refers to other amino acids having codons ending with a "C”. These form a CpG dinucleotide .if. followed by an Ala, Val or Glu codon (all of which begin with "G”) .
  • TTA for Leu is disfavored, but not prohibited.
  • a gene can be altered, without affecting the sequence of the encoded polypeptide, to reduce the number of CpGs to zero.
  • the gene is more preferably modified so that 1% to 3% of the dinucleotides are CpG.
  • CCGG Mspl/Hpall sites
  • a further consideration in designing the CpG-depleted marker gene is that one preferably should avoid creation of RNA splice sites. Consensus sequences for splicing donor and acceptor sites are given in Padgett, et al., Ann. Rev. Biochem. , 55:1119-50 (1986) . Otherwise, some mRNAs will be incorrectly spliced and may therefore be translated into a nonfunctional protein or a protein of different antigenic characteristics. ' The sequence AGGT is particularly undesirable as it is the predominant splice donor site; AGGC (a splice donor site) and AGG (a splice acceptor) should also be avoided if possible.
  • the desired CpG-depleted marker gene may be prepared entirely synthetically, i.e., using DNA synthesizer apparatus. See Worall and Connolly, J. Biol. Chem., 265:21889-95 (1990). (Typically, the double stranded DNA will be subdivided into overlapping single stranded oligonucleotide segments. These will be synthesized separately, then ligated and annealed to form the desired DNA duplex.) However, if the marker gene is very large, it may be more desirable to eliminate unwanted CpGs through mutagenesis, e.g., cassette mutagenesis, of the wild- type gene. The individual cassettes may, of course, be prepared synthetically as described above.
  • the invention is not limited to any particular method of preparing the CpG- depleted gene.
  • mutant is not intended to indicate that the wild-type gene is obtained first, and then altered. It includes even a wholly synthetic gene, provided that gene differs by at least one base pair from the naturally occurring gene which is closest in sequence to the mutant marker gene.
  • a CpG-depleted mutant gene 0 is one having at least one fewer CpG than the naturally occurring gene which has the greatest sequence similarity to the CpG- depleted gene.
  • the engineered structural sequence of the marker gene will be operably linked to regulatory sequences which are functional 5 in the cells in which the selectable or screenable phenotype conferred by the marker gene is to be looked for.
  • the most important of these regulatory sequences are the promoters.
  • the transcription of the coding strand of the gene is accomplished by DNA-directed RNA polymerase, which binds to the promoter 0 region.
  • Promoters may contain regulatory elements which render transcription tissue- or developmentally-specific, or which make transcription regulatable by inducer or repressor molecules.
  • the promoter may be constitutive, inducible or repressible; the choice will depend 5 on the character of the polypeptide encoded by the marker gene. •
  • mice A great variety of promoters have been used to drive expression of unrelated genes in transgenic animals.
  • mouse metallothionein (MT) human MT
  • mouse serum amyloid (SAA) mouse myc
  • mouse alpha2 mouse alpha2
  • mouse H-2K (class I MHC) , viral thymidine kinase, Rous sarcoma virus LTR, mouse iriammary tumor virus LTR, rat elastase, mouse albumin, mouse transferrin, human growth hormone releasing factor, mouse alphaA-crystallin, mouse beta- globin, mouse IgH and mouse amylase promoters. See Palmiter and
  • the promoter used should be one which is not tissue- specific.
  • a preferred promoter for driving expression of a marker gene is the beta-actin promoter, which, unlike the alpha-actin promoter, drives a gene whose expression is believed to be ubiquitous.
  • beta-actin promoter For the sequence of the beta-actin promoter from -2011, see Miyamoto, Nucleic Acids Res. , 15: 9095 (1987).
  • Other preferred ubiquitous promoters include the various tRNA promoters, the ribosomal RNA promoter, the ribosomal protein promoter, and the histone promoter.
  • methionyl tRNA promoter see Nucleic Acids Res., 12:1101-15 (1984).
  • the present invention extends, however, to the use of tissue-specific promoters as well.
  • the terminator (polyA addition site) sequence may be the endogenous terminator sequence of the marker gene, or it may be a foreign terminator, such as the terminator of the SV40 early gene or of the bovine growth hormone gene.
  • the ribosomal binding site may be the endogenous ribosomal binding site, or one which provides increased translational efficiency, such as the Kozak sequence.
  • Enhancer sequences may be used to increase expression, or to limit it to particular tissues., developmental stages, etc.
  • a regulatory element of interest appears in the first intron of the beta-actin gene. It is believed to act as an up-regulator of transcription in a non- tissue specific manner.
  • the marker gene and its associated regulatory sequences hereinafter referred to as the transgene, must be introduced into the cells of a host animal.
  • the target species is a vertebrate
  • the host animal is also, preferably, a vertebrate.
  • a suitable host animal is dependent on (a) its genetic and metabolic similarity to the target animal, and (b) the time and expense involved in producing and maintaining the transgenic animals.
  • Preferred host animals include, among the mammals, mice, rats, rabbits, hamsters and pigs, and among other vertebrates, transgenic fish.
  • Pigs are of interest since the anatomy of the pig (including the skin) is very similar to the human. Directing transgenic expression to the skin of pigs would create a useful model for the testing of cosmetics.
  • Fish may have an advantage in that various species of fish exhibit desirable characteristics relating to their use as laboratory animals. " In fact, fish have a long history of performing in this capacity. They have played a critical role in the development of environmental biology, - embryology, endocrinology, neurobiology and other areas. Research in fish has established much of our basic knowledge of membrane transport systems at the molecular level. Transgenic examples of at least 10 different species of fish have been produced. Several mammalian promoters have been shown to function in fish (1) . See Chen, Thomas T. and Powers, Dennis A., Transgenic fish, Trends in Biotechnology, Vol. 8, No. 8, 1990, pp. 209-215.
  • SV40 early promoter include the Rous sarcoma virus LTR promoter, the mouse metallothionein promoter, the flounder luciferase promoter and the flounder alpha fetoprotein promoter. It is further believed that the cytomegalovirus promoter and phosphoenolpyruvate carboxykinase (PEPCK) promoters would be functional in fish. In general, promoters of piscine genes, genes of viruses which infect fish, and genes which are strongly conserved among the vertebrates are likely to be functional in fish.
  • PEPCK phosphoenolpyruvate carboxykinase
  • Useful marker genes for fish models include the chloramphenicol acetyltransferase gene and the luciferase gene.
  • Stuart, et al., Development, 109:577-584 (1990) describes an assay for expression of a CAT transgene.
  • Assays for other genes transferred to fish, including various growth hormoned, the E. coli beta-galactosidase (lacZ) gene and the E. coli hygromycin resistance gene have been reported in the references cited in Table 1 of Chen, et al., and of course assays for expression of still more genes may be adapted from piscine systems.
  • the zebrafish (Brachydanio rerio) has been used to produce stable lines that exhibit reproducible patterns of transgene expression. See Stuart, Gary W.. , et al., Stable lines of transgenic zebrafish exhibit reproducable patterns of transgene expression, Development 109, 577-584, 1990. They are much less expensive to buy and raise than any mammalian species. They are extremely fecund, oviparous and are externally fertilized. Because of these factors, it is much less expensive and technically less complicated to perform gene transfer procedures on them. Their eggs are transparent and embryonic development occurs at a much faster rate than in the mouse. Large scale production of homozygous diploid zebrafish can be obtained in a reproducible and relatively simple manner. See Streisinger, G. , Walker, C., Dower, N. , Knauber, D., Singer, F. , Nature 291, 293, 1981.
  • Certain enzymes are known to play a role in the conversion of promutagens into mutagens.
  • Host animals may be selected, on a species and/or individual level, to provide a level of activity of these enzymes which is comparable to (or if desired to increase the margin of safety, higher than) that of the target animal of interest. If a particular species of animal, such as a mouse, is deficient in a particular enzyme of this type, it may be modified, by crossbreeding or genetic engineering, to provide
  • a transgenic animal may be produced that features a P450 enzyme missing in the mouse, or homologous recombination may be used to replace , it with a human counterpart' or to insert a stronger promoter upstream of a gene encoding such an enzyme.
  • this background level of mutation is low, e.g., less than 10 *5 to 10" 6 .
  • the natural environment of an animal may make it better suited for testing certain scenarios of chemical exposure.
  • waterborne chemical are preferably tested using transgenic fish (or amphibia or aquatic mammals) .
  • an animal is particularly sensitive to mutagens, it may be useful in detecting less potent mutagens.
  • a final issue is the economic importance of the animal.
  • a chemical which has a detrimental effect on an economically important animal may be rejected even if it does not have a serious adverse effect on humans. This could be the case with, for example, honey bees, or with fish.
  • the laboratory mouse has been the most popular host animal for use in the development of transgenic animals, as there are numerous strains available. Mice are, of course, the most widely available laboratory animal, and many strains are available. See Genetic Variants and Strains of the Laboratory Mouse (Gustav Fischer Verlag, 1981) . However, there are no substantial restrictions on the use of other laboratory or livestock species in such work. Among the higher mammals, pigs are preferred, and fish offer an interesting alternative to mammalian subjects.
  • DNA may be introduced into host cells by microi ⁇ jection, electroporation, infection, and other mechanisms such as lipofection and cell receptor-mediated transfer. While the DNA may plainly contain bacterial genes, procaryotic vector DNA (more particularly any prokaryotic replicon) should be removed before the transgene is introduced into the host cell(s) to be developed into a transgenic animal.
  • transgenic animals The most common technique for the production of transgenic animals involves the microinjection of the transgene into the pronucleus of fertilized eggs. Because integration usually accompanies DNA replication, about 70% of the transgenic mice carry the transgenes in all of their cells, including the germ cells. In the remaining 30%, integration apparently occurs after one or more rounds of replication, hence, the transgene is found in only a fraction of the cells. These mice usually exhibit the same degree of mosaicism in somatic and germ cells, but in some mice the germ cells may totally lack the transgene. In the latter case, the mice will be unable to transmit the transgene to their progeny.
  • One of the requirements for successful pronuclear microinjection is the ability to locate the pronucleus.
  • Transgenes may also be incorporated into the host cell genome by microinjection of DNA into the cytoplasm of fertilized or unfertilized eggs, into the nuclei of two-cell embryos, or into the blastocoel cavity. Mosaicism is more prevalent with these approaches.
  • microinjection alternatives include electroporation, liposome-mediated entry, and particle gun bombardment.
  • Preimplantation embryos may also be infected with retroviruses engineered to carry the transgene. This method has found particular favor for the production of transgenic birds.
  • Still another method for the production of transgenic animals is to introduce the transgene, on-a suitable vector, into totipotent teratocarcinoma or embryonic stem cells and then incorporate these cells into embryos.
  • transgenic animals are produced, they (or their transgenic progeny) are exposed to the suspect chemical.
  • the exposure may be by ingestion, inhalation, injection, or skin contact.
  • the dosage employed may be one comparable to that experienced by the target species in the environment of interest, or it may be a higher dose, in order to provide a margin of safety.
  • the animals are examined to determine whether the marker gene has been mutated.
  • the marker gene confers a phenotype which can be detected without killing the animal, e.g., one which may be detected by in vivo imaging means.
  • In vivo imaging means known in medicine include CAT, PET, NMR and MRI.
  • the transgene or its expression product must have a characterizing feature which is recognizable by a detectably labeled homing agent.
  • monoclonal antibodies may be prepared which bind a wild-type polypeptide, in preference to the mutant polypeptide encoded by the marker gene. These antibodies may be detectably labeled and injected into the animal. If the epitopes for these antibodies are reestablished by specific reverse mutation, by mutation, these antibodies may be localized by scintigraphic means known in the art. (A forward assay can also be envisioned, but is less desirable because of the increased background.)
  • Certain cells may, of course, be removed without killing the transgenic animal. These include blood cells, skin cells, mucosal cells, etc. Such cells may be removed and examined as described below.
  • this method while permitting the monitoring of the development of the mutagenic effect of the chemical in certain tissues over time, does not provide information as to mutagenesis of the marker gene in all tissues and organs. Therefore, in another and preferred embodiment, the animal is sacrificed so that all of its tissues and organs of interest may be examined for mutation of the transgene. In this embodiment, it is not strictly necessary that the animal have expressed the marker gene. However, it is preferable that the animal express the marker gene, since mutation rates may be different for expressed and unexpressed genes. Mellon, et al., PNAS, 83:8878-82 (1986).
  • transgenic mice which are homozygous for lad are mated with transgenic mice which are homozygous for lacZ under lacO control.
  • the progeny are hemizygous for a single copy of lad and for one or two copies of lacZ.
  • the progeny animals are exposed to the potential mutagen. If the lad gene is mutated, the cells of the progeny animal will stain blue since lacZ gene is then derepressed. (Having more than one copy of the lad gene is undesirable, since then both copies must be mutated in order to derepress the lacZ gene.)
  • a variety of other phenotypic characteristics could be used to identify cells containing a mutagenized form of the marker gene. These include antibody sensitivity or resistance, antigenicity, etc. (See discussion of marker genes above.)
  • the marker gene may be recovered from the genomic DNA of the transgenic animal.
  • a variety of techniques are known for rescue of a foreign gene from genomic DNA. These include rescue of lambda proviruses, plasmid rescue, and rescue of filamentous phage DNA.
  • One method is the use, ' as previously discussed, of a lambda packaging extract.
  • mutations may be detected in any of several ways. First, as the marker gene confers a selectable or screenable phenotype, the recovered marker gene may be cloned into a suitable "assay" cell, such as a bacterial cell, and the transformed cells may then be exposed to selection or screening conditions.
  • the marker gene must be expressible in the assay cells, and therefore must be operably linked (either originally, or as a result of further manipulation) to a promoter functional in those cells. This procedure will detect forward and back mutations, but not silent or neutral mutations. Silent and neutral mutations may be screened for by extracting genomic DNA and hybridizing it to a panel of oligonucleotide probes, each directed against a different locus of the marker gene, under stringent conditions. The failure of one of- these probes to hybridize is then indicative of the presence of a mutation.
  • transgenic animals in mutagenic assays also allows one to determine whether a promutagen or its metabolic products can cross the placenta or the blood-brain barriers.
  • Plasmid pCMVlad (5.5Kb) (Brown, et al., Cell, 49:603-12, 1987; Figge, et al., Cell, 52:713-22, 1988), a source of the lad gene, was digested with EcoRI, and the resulting 1.1Kb fragment was cloned into the EcoRI site of plasmid pBSK+ (2.9Kb) (Stratagene) , creating the plasmid pBSK lad (4.0Kb) . No promoter is operably linked to the lad gene in pBSK lad.
  • the 0-actin promoter was excised from the plasmid pHj8Apr-l (6.6Kb) (Gunning, et al., Proc. Nat. Acad. Sci. USA, 84:4831-35, 1987) by restriction with Hindlll and BamHI, and this 4.3Kb fragment was ligated with a 1.1 Kb fragment obtained by Ba ⁇ iHI/Hindlll digestion of pBSSKlad to obtain pH/Jlad (7.7Kb), in which the lad gene is under the transcriptional control of the j8-actin promoter.
  • the hybrid gene was modified to further encode the heptapeptide (PKKKRKV) nuclear location signal from SV40 large T antigen.
  • Plasmid pHjSlacI (7.7 Kb) was cut with Hindlll and BamHI, thereby excising the 3' untranslated flanking region of the lad gene.
  • the remaining 6.6 Kb fragment was ligated with a l.l Kb fragment obtained by digestion of plasmid pSZN5 (a.k.a.
  • pMTlacINLS a derivative of pMTlad (Brown, et al., Cell, 49:603-12, 1987) with Hindlll and Bglll, thereby producing the new plasmid pHjSlacINLS (7.7 Kb) .
  • Plasmid tkneo was cut with BamHI and Hindlll, releasing a 2 Kb fragment. Both ends of this fragment were then blunt-ended. Plasmid HSL mutants pSAM was cut with EcoRI, yielding a 2.7 Kb fragment. This, too, was blunt-ended. The two blunt-ended fragments were then ligated to obtain plasmid HSLmutants-neo. This was cut with Seal to Obtain a 2.6 Kb fragment.
  • Plasmid pHjSlacINLS was linearized with Sspl, and ligated to the neo-bearing Seal fragment to obtain pHSlacINLSneo (10.7 Kb) .
  • Plasmid pHblNB was prepared by cutting pHblacI with HindHI/BamHI, and ligating it with a 1.1 Hindlll/BamHI fragment from obtained by cutting pSZN5 with Bglll, blunt ending the Bglll ends, cutting again with Hindlll, and attaching a BamHI linker.
  • Transgenic mice were produced substantially according to the following standard protocol. (For further details, see Chandrashekar, et al., Neuroendocrine Research Methods and Functions in Transgenic Mice, in Greenstein, D.B., ed. , Vol. 1, Chap 15, Neuroendocrine Research Methods. 315-336 (Howard Academic Pub., London: 1991) .
  • Embryos from B 6 SJL F t female mice bred to males of the same strain are used in our laboratory because they culture well in our hands and are favorable for microinjectionbecause they have little cytoplasmic pigmentation.
  • the embryo donor B 6 SJL females are superovulated with 5 I.U.
  • mice pregnant mares' serum gonadotropin (PMS) at 12:00 noon three days prior to embryo collection. Forty-eight hours later at 12:00 noon, the ovulation of these mice is synchronized by injection of 5 I.U. of human chorionic gonadotropin (HCG) and embryos are collected at 9:00 am the following morning from the ampula of the oviducts of the embryo donors following sacrifice by cervical fracture. The collected embryos are treated with bovine testis hyaluronidase to remove cumulus cells, washed five times, and incubated under 90% N 2 , 5%0 2 , 5% C0 2 at 37°C in Brinster's medium until further use.
  • HCG human chorionic gonadotropin
  • Plasmid pHbLacI was digested with Sspl and BamHI to remove all procaryotic vector sequences, and 25 ⁇ l of fragments (concentration 25 ng/ ⁇ l) were microinjected into the male pronucleus of the collected embryos. Microinjection is carried out using two Leitz micromanipulators controlling a suction holding pipette and an l ⁇ m injection pipette.
  • the holding pipette is connected via tubing to a 500 ⁇ l threaded plunger Hamilton syringe; the injection pipette is connected via tubing to a microsyringe. In both cases the syringe and tubing are filled with light paraffin oil.
  • the injected embryos are transferred into the oviducts of white CD-I female mice previously bred to vasectomized males. These recipient females are selected by the presence of vaginal plugs on the morning the embryo microsurgery is performed. Ten injected embryos are transferred to each oviduct of each recipient. Approximately 20 days later, pups are born. When uninjected embryos are transferred as controls, the average litter size is 14. In our experience, 90 to 95% of recipients will give birth with an average litter size of 7 to 8 pups. Mice produced from microinjected eggs are weaned a month after birth. Segments of tails are analyzed by DNA hybridization analysis for the presence of the injected gene construct. In the instant experiment, the microinjected embryos were transplanted into the preimplantation uteri of nine pseudopregnant females. These females produced 63 pups, in eight litters.
  • Genomic DNA from the tails of several transgenic mice was digested with a restriction enzyme (BamHI, Bglll, EcoRI, Hindlll, Hpall, Mspl, NotI, PstI or Rsal) and characterized by Southern blotting.
  • the probe was prepared by digesting plasmid pCMVlad with EcoRV, which linearizes the plasmid, and then labeling the linearized pCMVlad with [ 32 P] dATP. The probe was hybridized to the blotted fragments at 42 deg. C.
  • lad cell line DNA (mouse NIH 3T3 or human fibrosarcoma HTD114 derivatives) was subjected to a similar analysis.
  • the Hpall and Mspl patterns were the same, indicating that lad was not methylated in cell line DNA.
  • mice 02.11.03 (female) (BCF1 background), 09.07 (female) (DBA/2J bkgd) and 09.03.03 (male) (129/SV bkgd) were sacrificed, and their liver, spleen, heart, testis/ovary (09.03.03 and 02.11.03 only), uterus, kidney andmuscle (02.11.03 and 09.03.03 only) tissues were removed and frozen in liquid nitrogen. Liver, testis/kidney and heart RNA was extracted by the acid phenol method.
  • a sensitive lad probe was made by PCR amplification of a lad template This was hybridized to the aforementioned RNAs.
  • Southern blots were prepared of lacI09 lineage DNAs to compare the degree of methylation of the lad gene in DBA/2J, 129/Sv and BALB/c X C3H backgrounds. There were no apparent differences in the first generation. In later generations, the lad gene was demethylated to some degree in the DBA/2J line but remained hypermethylated in the other two lines.
  • Figure 2 depicts a lad gene modified to reduce the number of CpG dinucleotides from 95 ( ⁇ 18%) to four ( ⁇ 0.8%).
  • the gene of Figure 2 is prepared by chemical synthesis of component oligonucleotides and their subsequent ligation and annealing to form the desired lad mutant.
  • the Lad gene was synthesized in segments by annealing double stranded oligonucleotides with overhanging, complementary ends of 10 nucleotides.
  • the following single stranded oligonucleotides were synthesized for use in assembling the lacImlRNL gene, including the Hindlll site, the Kozak RBS, the modified lad sequence, the three codon linker- and the seven codon NLS-encoding sequence. The numbering begins with the first base on the sense strand in Figure 2 (SEQ ID NO:3) .
  • the oligonucleotides a through t and a' through t' were synthesized on an Applied Biosystems 391 DNA synthesizer (PCR-MATE) per the vendors instructions.
  • the oligonucleotide a is complementary to a', b to b ! , c to c', etc.
  • the Lad gene was modified to remove all but 4 CpGs, 3 of which (positions 48, 606 and 1027) are part of Mspl/Hpall methylation diagnostic sites.
  • the modified Lad gene was synthesized in 2 halves.
  • the 5' half is bounded by a Hindlll site and Apal site; the 3' half by an Apal site and a BamHI site.
  • the double stranded oligos a/a, b/b" and c/c' were incubated together, allowed to anneal, and ligated with T4 DNA ligase.
  • the trimeric product was separated by agarose gel electrophoresis and recovered from the gel. The same procedure was followed with double stranded oligonucleotides d/d 1 , e/e', f/f, and g/g', and with h/h' , j/j 1 and k/k' .
  • the three trimeric products were incubated together, allowed to anneal via complementary overhanging ends, and ligated with T4 DNA ligase in the presence of Bluescript SK plasmid DNA (Stratagene) that had been digested with Hindlll and Apal.
  • the DNAs were used to transform E. coli XL1 cells, and transformed cells with plasmids containing inserts were identified by the absence of blue color development after staining with the chromogenic agent X-gal (Sigma) and IPTG
  • Plasmid DNAs from white colonies were isolated and tested for a 570bp insert representing the 5' half of the modified Lad gene by digestion with Hindlll and Apal and size fractionation. Inserts of the correct size were sequenced to ensure that no unwanted mutations were inadvertently introduced. The same procedure was used to synthesize, ligate and clone the oligonucleotides encoding the 3' end of the modified Lad gene.
  • the double stranded oligonucleotides were annealed, ligated and recovered in the following groupings: 1/1', m/m * , and n/n' ; o/o', p/p 1 , q/q' and r/r" ; s/s', and t/t ! .
  • the three oligonucleotide multimers were annealed together and ligated in the presence of Bluescript SK plasmid DNA (Stratagene) , and the DNA used to transform E. coli XL1 cells. Plasmids with inserts were identified as above, and correct insert size determined by Apal/BamHl digestion. Inserts were sequenced to ensure the absence of unwanted mutations.
  • the complete gene was assembled by digesting the plasmid containing the 5 ⁇ half with Hindlll and Apal and the plasmid containing the 5' end with Apal and BamHI.
  • the inserts were recovered and annealed and ligated in the presence of plasmid Bluescript SK digested with Hindlll and BamHI in a 3 way ligation.
  • white colonies were picked after staining with X-gal and IPTG induction.
  • Proper insert size (1.13kb) was established by digesting plasmids with Hindlll and BamHI, and the entire modified Lad gene was sequenced to ensure that it was correct.
  • the gene had a 5' Hindlll site; the beta actin promoter has a 3' Hindlll site and can readily be linked at that site.
  • the final construct is cloned in Bluescript SK+ (Stratagene) .
  • the resulting expression vector is then digested with with Sspl and BamHI to linearize the vector and remove procaryotic sequences, and the lad-bearing fragment is then microinjected in the male pronuclei of fertilized mouse eggs as previously described. Production of transgenic animals is then by the method set forth above. It is expected that, as a result of the CpG depletion, the engineered lad gene will be only weakly methylated, and therefore will be better expressed.
  • the E. coli gpt gene may be used.
  • 40 are CpGs.
  • the nucleotide sequence and position of CpGs are shown in Figure 3 (SEQ ID NO:5) .
  • Oligonucleotides containing the modified DNA sequences with absent or reduced CpGs are synthesized using an Applied Biosystems* 391 DNA synthesizer.
  • the modified sequences and oligonucleotides and endpoints are denoted in Figure 4 (SEQ ID NO:7) .
  • the strategy is to hybridize complementary oligonucleotides (e.g., a and a'; b and b' etc.) , to form double stranded oligos with 10 nucleotide overhangs.
  • the double stranded oligos a/a 1 , b/b' and c/c ? are incubated together allowing ends to anneal, ligated with T4 ligase, and the trimeric product is purified from an agarose gel.
  • the same procedure is followed for oligonucleotides d/d' and e/e', and for oligonucleotides f/f* and g/g' and h/h' .
  • the gel purified trimeric and dimeric products are incubated together to allow annealing in the presence of EcoRV-digested Bluescript SK
  • E. coli XL1 cells (Stratagene) are transformed with the product and unstained colonies are picked for analysis after chromogenic staining with X-gal and IPTG. Plasmids from white colonies are checked for proper sized inserts by cleavage with EcoRI and Hindlll, and plasmids with inserts of about 460bp are subjected to DNA sequencing to ensure that the sequence is correct and no unwanted mutations are present.
  • Pigs are one of the standard experimental models for humans in clinical studies.
  • Transgenic pigs may be produced by the following procedure, which uses commercial cross-bred sows and boars.
  • Parental stock may come from the Landrace, Dodge, Duroc and Hampshire breeds. About, 24 h after previous litters are weaned, sows used as zygote donors are induced to ovulate with 400 i.u. PMSG (i.ifi.)
  • Donor sows are artificially inseminated with 120 ml of fresh, extended semen 24 and 30 h after the onset of oestrus. (Recipient sows are synchronized in oestrus with the donor sows, but are not inseminated.) A mid-ventral laparatomy is performed on the donor animals and zygotes are flush from the oviducts with warm (37 deg. C.) modified BMOC-3 medium containing HES.
  • the zonae pellucidae of the recovered eggs are examined for the presence of spermatozoa.
  • One and two- cellzygotes are centrifuged at 10,000 xG to faciliate visualization of pronuclear and nuclear structures.
  • the zygotes are placed in cover-slip chambers in microdrops of modified BMOC-3 covered in silicone oil.
  • Microinjection of about 10 pi of DNA-containing soluation follows the procedure previously described for the mouse experiments.
  • A' midventral laparotomy is performed on the recipient sows and zygotes are inserted into the oviduct of animals identified by ovarian morphology as having ovulated.
  • Example 9 Production of Transgenic Fish
  • the zebrafish, Brachydanio rerio is a simple vertebrate with a number of desirable characteristics. Hundreds of eggs can be produced daily on a year round basis from a small number of 5 adult fish. Eggs can be fertilized in vitro; as in the frog, fertilization is external. Zebrafish embryos are optically transparent, so embryonic development can be monitored and cell types within the embryo identified. The fish develop rapidly, hatching from their chorions at 2 to 3 days post fertilization. Q The generation time is only 3 to 4 months.
  • Zebrafish are maintained in aquaria under conditions conducive to rearing, mating and spawning, e.g., 12-16 fish per tank, 28.5 * 1., 14h light/lOh dark cycle.
  • zebrafish care and maintenance see Streisinger, Nat. Cancer Inst. Monogr. 5 65:53-58 (1984) .
  • the embryos in embryo medium are placed on a depression slide and injected with the aid of a dissecting microscope and
  • the DNA solution is injected cytoplasmically through a continuously flowing micropipette, the flow rate may be controlled with pressurized air. Phenol red may be added to the solution to aid in estimating the volume injected.
  • DNA may be extracted from whole fish at 1-3 weeks of age by
  • the zebrafish is a small, laboratory- adapted vertebrate species which can be cared for more easily than most mammalian subjects.
  • a variety of mutations may be detected by studying their effects on a CpG-depleted marker gene in a zebrafish model.
  • the marker, or mutational target, gene express a detectable product. It may instead express a product that serves as a substrate for the product of a second (reporter) gene, or as a cofactor for the action of that product, or, as in this example, as a means of regulating the expression of the second gene.
  • the Lac repressor expressed by a lad target gene, may be used to extinguish expression of beta- galactosidase by the lacZ indicator gene.
  • One method of obtaining a lad/lacZ transgenic animal is by mating (a) a transgenic mouse homozygous for a single copy of the target transgene lad, and (b) a transgenic mouse homozygous for the indicator transgene, lacZ, which may be present in one or two copies.
  • Production of mouse line (a) is described in Example 2.
  • Production of mouseline (b) is set forth in Example 11.
  • the modified lad gene with reduced CpG and (preferably) mammalian codon usage is directed by the normal bacterial lad promoter, or an alternative prokaryotic promoter such as trp, and introduced into a lambda phage shuttle vector that has been previously described with the bacterial lad by Kohler, et al., Proc. Natl. Acad. Sci. USA 88:7958-62. 1991, and that includes the c subunit of lacZ, a jS-lactamase gene, and a ColEl replication origin, all flanked by the initiator and terminator halves of the Fl filamentous phage origin.
  • the vector is introduced into mice by pronuclear injection, and the transgenic mice are bred to homozygosity for the transgene.
  • the mice are exposed to mutagen and genomic DNA is subsequently prepared from selected tissues as previously described: Kohler, S.W. et al.. Nucleic Acids Res. 18_:3007-13, 1990; Kohler, S.W. et al., PNAS _3_8:7958- 62, 1991.
  • the shuttle vector is rescued from genomic DNA by packaging the shuttle vector DNA into infective ⁇ virions using an in vitro ⁇ packaging extract (Transpack from Stratagene Cloning Systems) , preadsorbing to E.
  • coli SCS-8 (Stratagene Cloning Systems) , mixing with top agar containing 2 mg of X-gal per ml of top agar and pouring onto assay plates with a bottom agar layer.
  • Rescued phage containing wild type lad will produce colorless plaques while rescued phage with mutant lad will produce blue plaques.
  • the ratio of blue to colorless plaques is indicative of the mutagenicity of the compound.
  • the DNA containing mutant lad can be excised from the lambda phage in vivo (Kohler, S.W. , Nucleic Acids Res. 16:7583- 7600, 1988) , and the mutant lad gene sequenced.
  • the modified lad gene with prokaryotic promoter is linked to a DNA sequence that is recognized by and binds to a specific protein or other substance.
  • a specific protein or other substance is the lac operator (lacO) which specifically binds the lac repressor with high affinity.
  • lacO lac operator
  • the lac operator is placed close to the lad gene, so as not to interfere with expression, and mice are rendered transgenic for this construct by pronuclear injection. After breeding mice to homozygosity for the transgene, the animals are exposed to he mutagenic environment.
  • the DNAs are isolated from selected organs and tissues of the exposed animal, and the DNA is digested with an enzyme that cleaves outside of both the operator sequence and the lad gene leaving intact DNA fragments containing both sequences.
  • lac repressor protein attached to magnetic beads.
  • the repressor binds the operator sequence, and the complex is separated from the remainder of the DNA by use of a magnet.
  • the separated fragments are cloned into a plasmid with an ampicillin-resistance marker and used to transform E. coli that constitutively express lacZ due to mutant or absent lad. Ampicillin resistant colonies containing mutant lad will stain blue with X-gal while colonies with wild-type lad will not.
  • bipartite detection system which is not limited to lad/lacZ
  • an "inhibitory" gene is the target gene
  • a reporter gene as the target gene (i.e., lacZ alone)
  • mutation in the target transgene is manifested as stained cells on an unstained background, whereas if lacZ were the target, mutation would appear as unstained cells on a stained background.
  • Example 11 Production of Transgenic Mice Expressing a Bacterial lacZ Gene
  • the constructs will be generated as described in Figure 6.
  • the promoterless ⁇ - galactosidase gene with 3' SV40 processing signals is bounded by Hindlll (5') and Xhol (3') sites in the Bluescript-based plasmid pLZ6.6 ( Figure 5B and 6A) .
  • the core of this sequence is an 18 bp palindrome that behaves as a mutant operator which binds lac repressor- about 8 times more tightly than the wild-type operator sequence Brown, et al., Cell, 4 :603-612, 1987.
  • the oligonucleotides will be hybridized and directionally cloned into the unique BamHl/Hindlll sites upstream of the lacZ gene ( Figure 6B) .
  • a 396 bp fragment extending from 6 nucleotides upstream of the APRT translation start codon (position -6) to position -402, encompassing the entire aprt promoter (Dush, et al., Nucleic Acids Res., 16:8509- 8524, 1988) and flanked by BamHI linkers, will be cloned immediately upstream of the operator sequence, and its orientation determined by the position of an asymmetrically located Smal site ( Figure 6C) .
  • a neo marker driven by an HSV tk promoter and flanked by Xhol sites will be inserted at the unique Xhol site in either orientation ( Figure 6D) for selection purposes, and the resultant plasmid designated pAPlacOZneo.
  • aprt promoter constructs complementary oligonucleotides encoding operator sequence with appropriate cohesive ends will be synthesized, and inserted at the sites indicated in Figure 7. Following conversion of the 3' end of the modified aprt promoter to a Hindlll site, the fragment will replace the aprt/lacO promoter construct in Figure 6D and will be inserted into the resulting BamHl/HindlH site immediately preceding the lacZ gene.
  • the system may be validated and optimized by testing various known and suspected mutagens and/or carcinogens.
  • N-nitrosoethyl urea a transplacental mutagen of the N-nitroso family of carcinogenic agents. It causes neurogenic tumors in a variety of species, including mice Rice, et al., Ann. N.Y. Acad. Sci., 381:274-289. 1982, and papillary lung tumors in the progeny of pregnant females exposed to the agent Rehm, et al., Cancer Res., 48.:148-160, 1988. Since there is no real precedent to follow, we will first expose fetal mice to NEU via i.p. injection of the mother with 0.1 mmol to 0.5 mmol NEU per Kg on gestational days 14, 16 and 18.
  • mice from different litters at 1 wk, 6 wk, 20 wk and 1 yr of age for each time and dose of NEU administration, which is equivalent to the number .used to unequivocally detect an association with lung tumors.
  • Analysis of stained sections should define which organ(s) and tissue(s) in progeny mice are most susceptible to mutation following i.p. ' administration of NEU to the mother. It will also define the gestational age at which the fetus is most susceptible to this agent, and will establish the presence or absence of a dose response relationship between the amount of NEU administered and the number of stained foci per organ than one detects.
  • a second class of carcinogen which may be tested is the aromatic amines, whose carcinogenic characteristics have been recognized since the turn of the century. These compounds have been widely used in industry, see Haley, et al. , Handbook of Carcinogens and Hazardous Substances, eds. M.C. Bowman, Marcel Dekker, Inc., New York, Basel, 1982. For example, the DuPont Company has screened workers exposed to jS- naphthylamine for the occurrence of bladder cancer Mason, et al., J. Occup. Med. , 2J[:1011-1016, 1986. Other studies (e.g.
  • target organs such as liver lack dividing cells so that a mutation in the lad gene will be manifested as only a single stained cell.
  • target organs such as liver lack dividing cells so that a mutation in the lad gene will be manifested as only a single stained cell.
  • immature animals at an age when mitotic division is still active in most organs like liver, larger foci will be evident due to a lad mutation in a progenitor cell and all of its progeny.
  • nursing mothers will be injected i.p. or i.v. immediately after birth of a litter of tester mice and offspring will be analyzed at 6 to 10 weeks of age.
  • mice will be exposed to increasing amounts of one of the above aromatic amines (each will be separately tested) .
  • Administration will be a single or multiple doses dispensed i.p., i.v. or orally (gavage) to again determine the target organ(s) and to ask whether they differ according to route of administration.
  • Mice will be sacrificed at times up to 8 weeks after the last administration and individual organs sectioned, stained and analyzed as above. We will begin with about 6 to 10 mice for each regimen of mutagen administration. This number may be increased if so required.
  • PCBs congenital poisoning in Taiwan in 1978 by cooking oil contaminated with thermally degraded PCBs.
  • Affected offspring of mothers who had ingested the contaminated rice-bran oil manifested a spectrum of congenital defects. It may be too early to tell whether or not these children will exhibit a higher than normal incidence of tumors.
  • PCB thermal derivatives are mutagenic to fetuses of pregnant mice that ingest these agents, and if so, whether or not 1) the affected tissue(s) is of ectodermal origin, as appears to be the case in man, and 2) whether or not tissues that incur mutations in the lad transgene later selectively give rise to tumors.
  • PCBs will be dissolved in cooking oil at about 100 pp . Chen, et al., Am. J. Int. Med., .5:133-145, 1984.
  • PCDF polychlorinated dibenzofuran
  • the offspring (after cooling) will be orally administered to pregnant mice carrying tester fetuses.
  • the offspring will be analyzed for teratological abnormalities and for organs and tissues that manifest lad mutations, as described before.
  • GTT TCT GCC AAA ACC AGG GAA AAA GTG GAA GCA GCC ATG GCA GAG CTG Val Ser Ala Lys Thr Arg Glu Lys Val Glu Ala Ala Met Ala Glu Leu 30 35 40 45

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Abstract

L'invention concerne un dosage basé sur un animal transgénique pour la détection de mutagènes ou carcinogènes et consistant à ajuster la fréquence dinucléotide CpG du transgène marqueur afin qu'elle soit similaire à celle des gènes natifs de l'animal hôte.
PCT/US1993/001676 1992-02-27 1993-02-26 Test de la mutagenicite a l'aide de genes rapporteurs avec frequences de methylation modifiees WO1993017123A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996012008A1 (fr) * 1994-10-13 1996-04-25 Merck & Co., Inc. Synthese de genes resistant a la methylase
WO1999062333A1 (fr) * 1998-05-31 1999-12-09 The University Of Georgia Research Foundation, Inc. Poisson transgenique derive d'un bacteriophage pour detecter des mutations
US6472583B1 (en) 1998-10-26 2002-10-29 The University Of Georgia Research Foundation, Inc. Plasmid-based mutation detection system in transgenic fish
EP1373297A4 (fr) * 2001-03-05 2005-09-21 Univ Virginia Systeme operateur-represseur lac

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0289121A2 (fr) * 1987-05-01 1988-11-02 Stratagene Test de mutagénèse utilisant des êtres non humains transgéniques porteurs des séquences d'ADN à essayer
WO1989005864A1 (fr) * 1987-12-15 1989-06-29 The Trustees Of Princeton University Systemes de test de transgeniques visant a detecter des mutagenes et des carcinogenes
EP0370813A2 (fr) * 1988-11-25 1990-05-30 Exemplar Corporation Dosage rapide par criblage de mutagénèse et tératogénèse

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Publication number Priority date Publication date Assignee Title
EP0289121A2 (fr) * 1987-05-01 1988-11-02 Stratagene Test de mutagénèse utilisant des êtres non humains transgéniques porteurs des séquences d'ADN à essayer
WO1989005864A1 (fr) * 1987-12-15 1989-06-29 The Trustees Of Princeton University Systemes de test de transgeniques visant a detecter des mutagenes et des carcinogenes
EP0370813A2 (fr) * 1988-11-25 1990-05-30 Exemplar Corporation Dosage rapide par criblage de mutagénèse et tératogénèse

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Title
NUC. ACIDS RES., Vol. 14, Suppl., issued 1986, MARUYAMA et al., "Codon Usage Tabulated from the GenBank Genetics Sequence Data", pages r151-r197. *
TIBTECH, Vol. 6, issued August 1988, ERNST, J.B., "Codon Usage and Gene Expression", pages 196-199. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996012008A1 (fr) * 1994-10-13 1996-04-25 Merck & Co., Inc. Synthese de genes resistant a la methylase
WO1999062333A1 (fr) * 1998-05-31 1999-12-09 The University Of Georgia Research Foundation, Inc. Poisson transgenique derive d'un bacteriophage pour detecter des mutations
US6307121B1 (en) 1998-05-31 2001-10-23 The University Of Georgia Research Foundation, Inc. Bacteriophage-based transgenic fish for mutation detection
US6472583B1 (en) 1998-10-26 2002-10-29 The University Of Georgia Research Foundation, Inc. Plasmid-based mutation detection system in transgenic fish
EP1373297A4 (fr) * 2001-03-05 2005-09-21 Univ Virginia Systeme operateur-represseur lac

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