WO1995007106A1 - Procede d'identification d'agents mutagenes au moyen de sperme de souris transgeniques - Google Patents

Procede d'identification d'agents mutagenes au moyen de sperme de souris transgeniques Download PDF

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WO1995007106A1
WO1995007106A1 PCT/US1994/009724 US9409724W WO9507106A1 WO 1995007106 A1 WO1995007106 A1 WO 1995007106A1 US 9409724 W US9409724 W US 9409724W WO 9507106 A1 WO9507106 A1 WO 9507106A1
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protamine
human
sperm
variant
mouse
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PCT/US1994/009724
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Andrew J. Wyrobek
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The Regents Of The University Of California
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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
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • 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
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • 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
    • 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

Definitions

  • the present invention relates generally to methods for detecting mutagen ⁇ .
  • the invention relates to methods for identifying agents which cause germinal and heritable mutations, using sperm from
  • transgenic animals carrying a heterologous sperm cell protein gene or a variant thereof.
  • Germinal mutations are mutations that originate
  • chromosomal abnormalities such as aneuploidy and structural changes in chromosomes.
  • Protamines are small, highly charged proteins that package the DNA of most vertebrate sperm in a condensed, genetically inactive state.
  • prota ine 1 PI
  • protamine 2 P2
  • the primary sequences for human protamines 1 and 2 have been determined. Balhorn et al., iotech ol . Appl . Biochem . (1987) .9:82- 88; Mckay et al., Eur. J . Biochem . (1986) 156:5-8; Mckay et al., Biosci . Rep . (1985) 5 . : 383-391.
  • PI is 50 a ino acids in length and the sequence differs markedly from
  • P2 occurs in two variant forms known as P2a and P2b.
  • P2a and P2b differ only by three amino acids—P2b having 54 amino acids and P2a including three additional amino acids at the amino terminus.
  • WO 91/19810 (published 12/26/91) ; WO 91/07489 (published 5/30/91) ; WO 91/04327 (published 4/4/91) ; WO 91/10741 (published 7/25/91) ; WO 91/08216 (published 6/13/91) .
  • the use of transgenic animals to detect germinal mutations has not heretofore been disclosed.
  • the present invention is based on the discovery of a system for rapidly and effectively detecting heritable mutations using sperm cells rather than whole animals.
  • mutagenic agents can be identified using sperm derived from transgenic animals.
  • the transgenic animal will either include a gene encoding a heterologous sperm cell protein, a gene encoding a mutein of the sperm cell protein or a variant gene incapable of transcription unless a mutation occurs. If the animal is exposed to a germinal mutagenic agent, mutations will occur in the sperm cell protein gene, thereby changing the specificity of the gene product and allowing detection of the same.
  • the subject invention is directed to a method for identifying a mutagenic agent capable of causing a germinal mutation.
  • the method comprises: (a) exposing a transgenic nonhuman male mammal that expresses a heterologous sperm cell protein to an agent suspected of being mutagenic;
  • the invention is directed to a method for identifying a mutagenic agent capable of causing a germinal mutation. The method comprises:
  • the invention is directed to a transgene comprising:
  • the invention is directed to plasmid pBmPl.hPl.
  • the invention is directed to transgenic mice and embryos transformed with these constructs.
  • Figure 1 depicts recombinant plasmid constructs containing the human protamine gene coding sequences and mouse protamine promoter sequences.
  • Figure 2 shows the relative fluorescence brightness as measured in a flow cyto eter of monoclonal antibody hup 1M reacted with human and mouse sperm nuclei.
  • Figure 3 shows the human PI cDNA sequence, the corresponding amino acid sequence and sites at which stop and nonsense codons (TAA, TAG and TGA) can be introduced by a single base change.
  • sperm cell protein any of the various proteins normally expressed in mammalian sperm cells. Such proteins include nuclear, extra-nuclear, acrosomal, surface, mitochondrial, mid-piece, principle piece and tail proteins. Examples of these proteins include but are not limited to protamines, spermine, spermidine, clupeine, any of the various histones, various sperm nulcear protein, among others.
  • the term is also meant to encompass variants of these proteins such as molecules having amino acid sequences substantially homologous to contiguous amino acid sequences of naturally occurring sperm cell proteins. Thus, the term includes both full-length, truncated and partial sequences, as well as mutant proteins having amino acid substitutions, insertions and deletions and precursor forms of the proteins, capable of expression in sperm of the transgenic animal.
  • protamine is meant any of the various known vertebrate protamines, including mammalian protamines such as canine, feline, equine, ovine, porcine, bovine, rodent, human and other primate protamines, among others.
  • mammalian protamines such as canine, feline, equine, ovine, porcine, bovine, rodent, human and other primate protamines, among others.
  • Particularly preferred protamines include human protamine PI and P2, as well as variants of PI and P2, including P2a and P2b.
  • protamine encompasses molecules having amino acid sequences substantially homologous to contiguous amino acid sequences of naturally occurring protamines and includes both full-length, truncated and partial sequences, as well as muteins having amino acid substitutions, insertions and deletions, capable of expression in sperm of the transgenic animal.
  • transgene is meant a recombinant DNA construct, including a heterologous gene encoding a sperm cell protein along with control elements (as described below) , which is capable of expression in vivo in a transgenic animal.
  • a "transgenic" embryo or animal is an embryo or animal, respectively, which includes a transgene encoding a heterologous sperm cell protein.
  • the organism will be stably transformed, that is, the genetic material will be integrated into the chromosome so that it is inherited by daughter cells through chromosome replication.
  • the term animal encompasses new-born as well as fully developed animals.
  • nonhuman mammalian animal any nonhuman member of the class mammalia, including, but not limited to, cats, dogs, rodents such as mice, rats and guinea pigs, rabbits, bovine, porcine and ovine (sheep and goats) species, and other wild or domestic animals, pets and laboratory animals.
  • heterologous or “foreign,” as they relate to nucleic acid sequences, denote sequences that are not normally associated with a region of a recombinant construct, and/or are not normally associated with a particular cell.
  • a “heterologous" region of a nucleic acid construct is an identifiable segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. Accordingly, when the heterologous region encodes a eucaryotic gene, the gene will usually be flanked by sequences that do not flank the eucaryotic gene in the nature.
  • heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene) .
  • a transgenic organism or sperm cell therefrom, bearing "heterologous” or “foreign” DNA would include DNA sequences not normally found in the organism or sperm cell. Allelic variation or naturally occurring mutational events do not give rise to foreign DNA, as used herein.
  • a "coding sequence” is a nucleotide sequence which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, cDNA from eucaryotic RNA, genomic DNA sequences from eucaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the coding sequence.
  • a "nucleotide sequence” can include, but is not limited to, procaryotic sequences, eucaryotic mRNA, cDNA from eucaryotic mRNA, genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
  • the term also captures sequences that include any of the known base analogs of DNA and RNA such as, but not limited to 4-acetylcytosine, 8-hydroxy-N6- methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxyl ethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo- uracil, 1-methylguanine, 1-methylinosine, 2 ,2-dimethyl- guanine, 2-methyladenine, 2-methylguanine, 3-methyl- cytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy- aminomethyl-2
  • 2-thiouracil 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2, 6-diaminopurine.
  • a transcription termination sequence will usually be located 3' to the coding sequence.
  • control sequences refers collectively to promoter sequences, riboso e binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the transcription and translation of a coding sequence in a host cell. Not all of these control sequences need always be present in a recombinant vector so long as the desired gene is capable of being transcribed and translated in vivo .
  • operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
  • the control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • a control sequence "directs the transcription" of a coding sequence in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.
  • transgenic animals which express heterologous sperm cell proteins.
  • the transgenic animal is exposed to the substance and the sperm collected and analyzed for the loss or acquisition of a particular phenotype conferred by the foreign gene.
  • exemplified herein is the production of transgenic mice expressing human protamine genes and variants of these genes. After exposure to a potential mutagen, sperm are collected from the mice and the sperm analyzed for reactivity with an antibody specific with the human protamine.
  • the sperm will either lose the ability to react with the antibody (e.g., if the animal has been transformed with a gene encoding a protein reactive with the antibody) or gain the ability to react with the antibody (e.g., if the animal has been transformed with a gene variant which is either inactivated or encodes a protein normally unreactive with the antibody but which, by virtue of mutation, becomes reactive therewith) .
  • Reactivity is conveniently monitored using a fluorescent tag and flow cytometric procedures. Accordingly, the present invention not only provides for an efficient method of detecting germinal mutations, but also allows for an assessment of the type of mutation caused by the agent in question.
  • Transgenic animals suitable for the determination of GEL mutations are animals which express a heterologous sperm cell protein prior to exposure to a mutagenic agent. Generally, these animals are homozygous for a single haploid expressed copy of the gene. Thus, sperm expressing the heterologous protein are detectable, e.g., by reaction with antibodies specific thereto, while sperm with GEL mutations will not bind the antibody.
  • monoclonal antibody hup IM is described herein which is specifically reactive with human protamine PI but lacks reactivity with human protamine variants or with protamines from other species.
  • monoclonal antibody hup IN also described below, reacts with both mouse and human PI.
  • sperm carrying GEL mutations will not react with hup IM but will bind with hup IN.
  • Transgenic animals for detecting BPS mutations can include genes which have been inactivated by the introduction of stop codons into their genomes.
  • Figure 3 shows potential positions where stop codons can be introduced into the protamine PI gene by single base pair changes. Additional variants can also be produced by introducing nonsense codons at specified points of the native coding sequence. For example, with respect to human protamine PI, such nonsense codons can be introduced at 11 of the first 20 amino acid positions by a single-base change (see Figure 3) .
  • Representative strategies for introducing such mutations at triplets coding for amino acid positions 9 and 15 of human protamine PI are shown in Tables 1 and 2, respectively.
  • base pair changes can be introduced into the gene encoding the sperm cell protein of interest such ' that a variant protein is expressed.
  • Such variants may impart differing specificity to the molecule, or impart differing immunogenic characteristics thereto, such that the molecules can be distinguished from the native sperm cell proteins using assays as described below.
  • Transgenic animals for the detection of agents causing BPS mutations will generally have hundreds of copies of inactivated heterologous genes incorporated into their genomes in order to increase the number of mutable targets.
  • the majority of sperm from such animals will not react with antibodies specific for the heterologous ⁇ perm cell protein, while sperm in which BPS mutations have reverted due to exposure with a mutagenic agent will bind the antibody.
  • antibody hup IM described herein, will be useful for detecting these mutations since the hup IM antibodies will only be reactive when genes have been mutated by the presence of the mutagenic agent.
  • Table 3 The general strategy for assessing GEL and BPS mutations, is shown below in Table 3. Table 3
  • protamines 1 and 2 are conveniently used because they comprise the major sperm nuclear protein which coats and packages the entire surface of sperm DNA.
  • the gene sequences of various protamines are known. See, e.g., Domenjoud et al., Genomic ⁇ (1990) .8:127-133; Lee et al., Nucleic Acids Res , (1987) 15:7639.
  • sequences, variants thereof, as well as other sequences encoding ⁇ perm cell protein ⁇ are u ⁇ ed to construct recombinant vectors for transforming nonhuman, mammalian animals.
  • nucleic acid sequences for use in vectors for creating the transgenic animals can be isolated from cells and ti ⁇ ues containing the same, using standard techniques, such as phenol extraction. See, e.g., Sa brook et al., supra , for a description of techniques used to isolate DNA.
  • DNA encoding the desired sperm cell protein can be isolated directly from sperm cells.
  • the gene sequence if known, can be generated synthetically, using standard modes of polynucleotide ⁇ ynthesi ⁇ , well known in the art.
  • oligonucleotides are prepared by either the phosphotriester method as described by Edge et al., Nature (supra ) and Duckworth et al., Nuc . Acids Res . (1981) 9 . : 1691, or the phosphoramidite method a ⁇ de ⁇ cribed by Beaucage, S.L., and Caruthers, M.H., Tet . Letts .
  • a particularly convenient method for obtaining nucleic acid for use in the present invention is by recombinant means.
  • genes encoding the subject sperm cell proteins can be identified by constructing gene libraries, using the resulting clone ⁇ to transform an appropriate host cell, and pooling and screening individual colonies using polyclonal serum or monoclonal antibodies to the desired protein.
  • the gene sequence of the desired protein can also be obtained using the following general technique.
  • the desired sperm cell protein can be isolated from, for example, sperm cells containing the same. This is generally accomplished by first preparing a crude extract which lacks cellular components and several extraneous proteins.
  • the desired proteins can then be further purified, i.e., by column chromatography, HPLC, immunoadsorbent technique ⁇ or other conventional methods well known in the art. See, e.g., Balhorn et al., Arch . Biochem . Biophys . (1992) 296:384-393 , for a description of procedures for isolating mammalian protamine from semen.
  • amino acid sequences can be determined from the purified proteins by repetitive cycles of Edman degradation, followed by amino acid analysis by HPLC. Other methods of amino acid sequencing are also known in the art.
  • oligonucleotide probes which contain the codons for a portion of the determined amino acid sequences can be prepared and used to screen DNA libraries, for example, testicular cDNA libraries, for genes encoding the subject proteins.
  • the basic strategie ⁇ for preparing oligonucleotide probes and DNA libraries, as well as their screening by nucleic acid hybridization, are well known to those of ordinary skill in the art. See, e.g., DNA Cloning: Vol. I, supra ; Nucleic Acid Hybridization , supra ; Oligonucleotide Synthesis , supra ; Sa brook, et al. , supra .
  • the selected oligonucleotide probes are labeled with a marker, such as a radionucleotide or biotin, using standard procedures.
  • the labeled set of probe ⁇ is then used in the screening step, which consi ⁇ ts of allowing the single-stranded probe to hybridize to isolated ssDNA from the library, according to standard techniques.
  • the selection of the appropriate conditions i ⁇ within the skill of the art. See, generally, Nucleic Acid hybridization , supra .
  • Once a clone from the screened library has been identified by positive hybridization, it can be confirmed by restriction enzyme analysis and DNA sequencing that the particular library insert contains a gene for the desired protein.
  • the desired DNA sequence can then be cloned into a cloning vector and further used.
  • cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice.
  • Ligations to other sequences are performed using standard procedures. For example, ligations can be accomplished in 20 mM Tris-Cl pH 7.5, 10 M MgCl , 10 mM dithiothreitol (DTT) , 33 ⁇ g/ l BSA, 10 mM-50 mM NaCl, and either 40 ⁇ M ATP, 0.01-0.02 (Weis ⁇ ) units T4 DNA ligase at 0°C (for "sticky end” ligation) or 1 mM ATP, 0.3-0.6 (weiss) units T4 DNA ligase at 14°C (for "blunt end” ligation) . Intermolecular "sticky end” ligations are usually performed at 30-100 ug/ml total DNA concentrations (5-100 nM total end concentration) .
  • the gene can be placed under the control of a promoter, ribosome binding site and, optionally, an operator (collectively referred to herein as "control" elements) , ⁇ o that the gene ⁇ equence encoding the de ⁇ ired protein is transcribed into RNA in the host cell transformed by a vector containing this expression construct.
  • control elements collectively referred to herein as "control" elements
  • the coding sequence may or may not contain a signal peptide or leader sequence.
  • regulatory elements will depend on the system desired. For example, if the host's endogenous transcription and translation machinery will be used to expres ⁇ the proteins, control elements compatible with the particular host will be utilized.
  • promoters for use in mammalian sy ⁇ tem ⁇ are known in the art and include, but are not limited to promoters derived from SV40, CMV, HSV, RSV, MMTV, T7, T3 , among others.
  • i ⁇ the mouse protamine promoter to drive the expression of the human protamine gene and specifically, the mouse PI promoter in combination with the human PI coding sequence.
  • 5' and 3' UTR and termination sequences may also be present that are derived from the mouse and human protamine genes.
  • Various constructs containing these elements are shown in Figure 1 and are described in the Examples and in Table 4. Any combination of these elements, resulting in viable embryos with the ability to expres ⁇ the human protamine gene, will find use with the present invention.
  • regulatory sequences which allow for regulation of the expres ⁇ ion of the protein sequences.
  • Regulatory ⁇ equences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Other types of regulatory elements may also be pre ⁇ ent in the vector, for example, enhancer sequences.
  • An expres ⁇ ion vector is constructed so that the particular coding sequence i ⁇ located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the "control" of the control sequences (i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence) .
  • control sequences i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence
  • Modification of the sequences encoding the particular protein of interest may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation, i.e., to maintain the reading frame.
  • control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector.
  • the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.
  • the sperm cell protein gene can be inactivated such that expression only occurs when the gene is mutated into a form capable of expression. These mutants are particularly useful for the identification of utagens which cau ⁇ e ba ⁇ e pair substitution mutations ⁇ ince tran ⁇ ition or transversion mutations caused by the mutagenic agent can cause the gene to revert to the original sequence.
  • mutants described above may be prepared by the deletion of a portion of the ⁇ equence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence.
  • Techniques for producing the above-described mutants are known to those skilled in the art.
  • these mutations can be made to the native sequence using conventional techniques such as by preparing synthetic oligonucleotides including the mutations and inserting the mutated sequence into the gene of interest using restriction endonuclease digestion. (See, e.g., Kunkel, T.A. Proc . Natl . Acad . Sci . USA (1985) 8_2 .
  • the mutation ⁇ can be effected using a mismatched primer (generally 10-20 nucleotides in length) which hybridizes to the native nucleotide sequence (generally cDNA corresponding to the RNA sequence) , at a temperature below the melting temperature of the mismatched duplex.
  • the primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. Zoller and Smith, Methods Enzymol . (1983) 100:468.
  • Primer extension is effected using DNA polymerase, the product cloned and clone ⁇ containing the mutated DNA, derived by segregation of the primer extended strand, selected. Selection can be accomplished using the mutant primer as a hybridization probe.
  • the technique is also applicable for generating multiple point mutations. See, e.g., Dalbie-McFarland et al., Proc. Natl . Acad . Sci USA (1982) 79:6409.
  • the ⁇ e vector ⁇ can be used to produce transgenic animals.
  • a number of methods have been developed to produce small transgenic animals, such as mice, and any of these methods will find use herein.
  • the most commonly used technique involves the direct microinjection of DNA into the pronucleus of i ⁇ olated, fertilized oocytes.
  • the transformed, fertilized eggs are then implanted into a female ⁇ urrogate for ⁇ ub ⁇ equent development.
  • female mice are induced to superovulate, placed immediately with males for mating, oviducts removed and fertilized eggs obtained.
  • Eggs containing pronuclei are identified and microinjected with the DNA constructs containing the heterologou ⁇ ⁇ perm cell protein coding sequence.
  • the strategy for injecting the DNA constructs into the eggs will vary depending on the particular construct used. For example, if the construct is one which encodes a mutant sperm cell protein gene for use in the detection of base pair substitution mutation ⁇ , pronuclei are injected with high concentrations of the recombinant construct ⁇ to produce animals with a high copy number of the inactivated gene, thereby increasing the number of mutable targets. (See, e.g., Gordon, J.W. , and F.H. Ruddle Gene (1985) 13 . : 121 for a description of transgenic mice with hundreds of integrated copies.)
  • transgenic animals include infection of embryos with viral vectors, particularly cloned retroviruse ⁇ , which include the gene encoding the de ⁇ ired ⁇ perm cell protein. Recombinant viral vectors are ea ⁇ ily introduced into cultured preimplantation embryo ⁇ which have been denuded of their zona pellucida.
  • the embryos are then returned to a foster mother, and when born, can be analyzed for the presence or absence of the sperm cell protein.
  • Methods for introducing foreign genes into animal embryos are well known and discu ⁇ ed in e.g., Jaeni ⁇ ch, R.
  • Transgenic animals can also be created via gene transfer using embryonic stem cells. Genes can be introduced into such cells by DNA tran ⁇ fection or by retrovirus-mediated transduction, and the cell clones selected for the presence of foreign DNA. See, e.g.,
  • Sperm cells can also be used as vectors for introducing foreign DNA into eggs.
  • foreign- DNA bearing pla ⁇ ids can be mixed with sperm cells in solution and mouse eggs sub ⁇ equently fertilized with the transformed sperm. See, e.g., Lavitrano et al., Cell (1989) 5_2:717-723.
  • Heterologous DNA can also be introduced into sperm using electroporation, a technique whereby cell membranes are momentarily made porous by an electric field to allow the infusion of macromolecules.
  • electroporation a technique whereby cell membranes are momentarily made porous by an electric field to allow the infusion of macromolecules.
  • Liposome mediated delivery of DNA into ⁇ perm cell ⁇ for use in ⁇ ub ⁇ equent fertilization can al ⁇ o be u ⁇ ed.
  • Bachiller et al., Mol . Repro . and Dev . (1991) 30: 194-200 The pre ⁇ ence of the foreign DNA in an embryo or animal, created using any of the above techniques, can be confirmed using any of various methods.
  • nucleic acid can be isolated and amplified using the polymerase chain reaction, as described by Saiki et al., Science (1985) 230: 1350-1354.
  • the preparation can be probed with sequences derived from the gene of interest in standard Southern blot experiments.
  • Western blot analysi ⁇ can be performed by isolating protein from the transformed tissue and reacting the same with antiserum, a ⁇ well a ⁇ monoclonal or polyclonal antibodie ⁇ rai ⁇ ed again ⁇ t the protein of interest. Histological analysi ⁇ of tran ⁇ formed tissues can also be performed using antibodies which recognize one or more epitopes of the desired protein.
  • in situ hybridization techniques can be used to locate integrated DNA in transformed embryos.
  • mice are bred to homozygosity.
  • the ⁇ pontaneou ⁇ mutation rate ⁇ can be determined for each con ⁇ truct u ⁇ ing flow cytometry.
  • the mutagen sensitivity of the animal lines can be determined and compared using ethylnitrosourea, a potent point mutagen in spermatogonial ⁇ tem cells in animals.
  • the transgenic animal can be used to screen potential mutagenic agents.
  • the animal including the heterologous sperm cell protein is exposed to the potential mutagen, the method of exposure depending on the substance being tested. For example, some substances will require injection and others inhalation or topical application. The proper route of exposure, doses and length of exposure will be readily determined by one of skill in the art.
  • sperm cells are i ⁇ olated, the i ⁇ olation technique depending on the tran ⁇ genic animal in que ⁇ tion. For example, sperm can be collected using an artificial vagina or obtained directly from sperm storage organs such as the epididymis. Other techniques for collecting sperm from mammals are well known.
  • Sperm can then be assayed for the presence or absence of the sperm cell protein gene expression product, using any of several methods, limited only by the ability of the method to distingui ⁇ h between the heterologou ⁇ protein and any corresponding native protein that might be present in the transgenic organism. If the transgenic organism includes a gene encoding a non- variant heterologous protein (or a sequence nearly identical thereto, such that detection methods for the native sequence will also detect ⁇ light variants thereof) , sperm cells can be screened for the presence of this expression product. If assays fail to detect the product, this means that the sequence has mutated and the agent tested will be deemed to be mutagenic.
  • the sperm can be assayed for the presence of native sequence which will only be detected if mutations have occurred. Similarly, the sperm can be as ⁇ ayed for the presence of the variant sequence (i.e., with antibodies reactive with the variant sequence but not with the native sequence) and if the variant is not detected, this will be indicative of a mutation. Any as ⁇ ay can be u ⁇ ed to detect the pre ⁇ ence or absence of the sperm cell protein of interest, the only requirement being that the a ⁇ ay be able to di ⁇ tinguish between the heterologous sperm cell protein and any other sperm cell protein that might be present in the tran ⁇ genic animal.
  • Typical assays for use in the present system will employ i munodiagno ⁇ tic techniques.
  • Antibodies both polyclonal and monoclonal
  • raised to the sperm cell protein of interest can be used in immunoas ⁇ ays, such a ⁇ competition, direct reaction, or sandwich type assays, for identifying the presence or absence of the proteins by forming complexes therewith.
  • immunoas ⁇ ays such as a ⁇ competition, direct reaction, or sandwich type assays, for identifying the presence or absence of the proteins by forming complexes therewith.
  • Such assays include, but are not limited to, Western blots, agglutination tests, enzyme-labeled and mediated immunoassays, such as ELISA ⁇ , biotin/avidin type assays, radioimmunoassays, immunoelectrophoresis, immunoprecipitation, etc.
  • the reactions generally include detectable labels such a ⁇ fluore ⁇ cent, chemilumine ⁇ cent, radioactive, or enzymatic labels or dye molecules.
  • detectable labels such as a ⁇ fluore ⁇ cent, chemilumine ⁇ cent, radioactive, or enzymatic labels or dye molecules.
  • an immunoas ⁇ ay for detecting the ⁇ perm cell protein of intere ⁇ t will involve isolating and preparing the sperm sample, and then reacting it with antibodies directed against the protein of interest, under conditions that allow protein-antibody conjugates to form.
  • Solid supports can be used such as nitrocellulose, in membrane or microtiter well form; polyvinylchloride, in ⁇ heet ⁇ or microtiter wells; polystyrene latex, in beads or microtiter plates; polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads and the like.
  • the solid support i ⁇ first reacted with the ⁇ perm ⁇ ample, washed and then antibodies (either polyclonal or monoclonal) are applied.
  • a sandwich type format such as a sandwich ELISA as ⁇ ay
  • a commercially available anti-im unoglobulin e.g., anti-rabbit immunoglobulin conjugated to a detectable label, such as horseradi ⁇ h peroxidase, alkaline phosphatase or urease
  • a detectable label such as horseradi ⁇ h peroxidase, alkaline phosphatase or urease
  • An appropriate substrate is then used to develop a color reaction.
  • a "two antibody sandwich” assay can be used to detect the sperm cell proteins. In this technique, the solid support is reacted first with antibodies (either monoclonal or polyclonal) directed against the protein, washed and then exposed to the sperm sample.
  • Monoclonal or polyclonal antibodies are again added and the reaction visualized u ⁇ ing either a direct color reaction or u ⁇ ing a labeled ⁇ econd antibody, such as an anti-immunoglobulin labeled with horseradish peroxidase, alkaline phosphatase or urease.
  • a labeled ⁇ econd antibody such as an anti-immunoglobulin labeled with horseradish peroxidase, alkaline phosphatase or urease.
  • Assays can also be conducted in solution, such that the sperm cell protein and antibody thereto form complexes under precipitating conditions.
  • the precipitated co plexe ⁇ can then be separated from the test sample, for example, by centrifugation.
  • a particularly convenient method for use in detecting the sperm cell proteins of interest involves using fluorescently labeled monoclonal antibodies in combination with high speed flow cytometry. The method allows for the evaluation of large numbers of sperm cells in a very short period of time. Procedures for flow sorting fluorescently tagged ⁇ perm cells are known in the art and described in, e.g., Johnson et al., Bio. Reprod. (1989) 41:199-203. Antibodies for use in the as ⁇ ay ⁇ can be generated using conventional techniques. For example, if polyclonal antibodies are desired, a selected mammal, (e.g., mouse, rabbit, goat, horse, etc.) i ⁇ immunized with the particular ⁇ perm cell protein of intere ⁇ t.
  • a selected mammal e.g., mouse, rabbit, goat, horse, etc.
  • Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies i ⁇ used, the polyclonal antibodies can be purified by immunoaffinity chromatography, using known procedures. Monoclonal antibodies to the desired protein can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by using hybrido a technology is well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Ep ⁇ tein-Barr viru ⁇ . See, e.g., M. Schreier et al., Hybridoma Techniques (1980); Ham erling et al., Monoclonal Antibodies and T-cell
  • hup IM (described further below) is specific for human PI and does not react with human P2, mouse protamine, or the protamine of any other animal tested thus far.
  • Hup IN reacts with both mouse and human PI.
  • Hup A reacts with all mouse and human protamines. Therefore, hup IM is useful for identifying transgenic mice that express human protamine while the latter two antibodies can be used to confirm the presence of mouse protamine in sperm and to check for binding specificity.
  • the following examples are provided for illus- trative purposes only and are not meant to limit the scope of the present invention.
  • Monoclonal antibodies specific for human protamines and useful in the mutagen detection as ⁇ ays described herein, were produced as follows.
  • the sperm pellet was resuspended in Tris- ⁇ aline (0.01 M Tri ⁇ , 10 M dithiothreitol (DTT)), ultrasonicated briefly with a Branson sonicator (Shelton, CT) , and centrifuged at 4000 x g for 3 minutes.
  • the pellet wa ⁇ re ⁇ u ⁇ pended in 20 ml of 1% cetyltrimethylam onium bromide (CTAB) , 10 mM DTT, 10 mM Tri ⁇ , pH 8.0, ⁇ onicated briefly, and allowed to ⁇ it at 4°C for 30 minute ⁇ to di ⁇ olved the tail, acrosome and membranes surrounding the chromatin.
  • CTAB cetyltrimethylam onium bromide
  • the sperm nuclei were rinsed twice in Tris-saline containing 0.1% CTAB and stored at 4°C. After storage, brief ultrasonication wa ⁇ used to resuspend the nuclei.
  • the protamine Prior to chromatography, the protamine was reduced for 18 hours with a 50-fold exces ⁇ of DTT in 6 M GuCl, 2 mM ethylenediaminetetraacetic acid (EDTA) and dialyzed at lea ⁇ t 1.5 hours against 200 volumes of 10 mM HCl, 1 mM DTT.
  • the ion-pairing agent, trifluoroacetic acid (TFA) was added to a final concentration of 0.1% and the reduced protamine was chromatographed on a Nucleosil RP- C18 column (7.5 mm I.D. x 300 mm) .
  • Human PI was separated from P2a and P2b using a linear, 12 to 24% acetonitrile gradient in 0.1% TFA, run at a rate of 2 ml/min for 50 minutes. The fractions containing the protamines were frozen and lyophilized. HPLC peaks were identified by electrophoresis of the proteins in acid- urea gels as de ⁇ cribed in Stanker, L.H., et al., Hybridoma (1987) (5:293-303.
  • Serum was obtained 14 days after the last injection and tested for anti-protamine activity in an enzyme-linked immunosorption a ⁇ ay (ELISA) as de ⁇ cribed below, and the animal with the highe ⁇ t ⁇ erum titer selected for cell fusion. Seven days later, this animal was injected intravenously (IV) on each of three consecutive days with 50 ⁇ g of human protamine in ⁇ terile ⁇ aline. The next day, the ⁇ pleen was removed, minced, and placed in 25 mM Tris-HCl, 0.15 M NH 4 C1, pH 7.5, solution to lyse the red blood cells.
  • ELISA enzyme-linked immunosorption a ⁇ ay
  • SP2/0 myeloma cells were grown in M3 medium containing 2% fetal bovine serum. Splenocytes were fused with an equal number of SP2/0 myeloma cells a ⁇ described in Stanker et al. (1987) , supra . Cells were plated without a feeder layer in equal parts of M3 and Hana HB101 serum-free media (Hana Biological ⁇ , Berkeley, CA) containing 30 mM hypoxanthanine, 40 nM a inopterin, 30 ⁇ M thymidine, and 2% fetal bovine ⁇ erum. Fourteen day ⁇ after fusion, the yield of anti-protamine secreting hybridomas was determined by ELISA with whole human protamine as antigen.
  • the hybridomas were isolated as follows: For cell fusion #1, growing hybridomas were observed in approximately 90% of the wells 12 days after cell fusion. Culture fluid from 59 of 1920 wells gave positive responses when screened against whole human protamine. Cells from 7 wells showing the greatest activity were cloned by limiting dilution and used to generate monoclonal antibody-containing ascite ⁇ fluid. The ⁇ e 7 monoclonal antibodies were designated hup la, hup lb, hup lc, hup Id, hup le, hup A, and hup B. Cells from the remaining 52 wells were expanded in 24-well plates and frozen. The stable monoclonal antibody of particular interest was hup A.
  • Monoclonal antibodies hup la, hup lb, hub lc, hup Id and hup le showed strong activity to human PI, but little or no activity to the human P2a+P2b mixture.
  • Antibody hup IM reacted only with human PI while showing no binding specificity for P2a+P2b or for mouse protamine.
  • Antibody hup 2b reacted specifically with human P2a+P2b.
  • a human testicular cDNA library in lambda gtll and a radiolabeled 40-mer oligonucleotide probe were used to clone human PI cDNA.
  • a complete sequence of the human PI cDNA has been obtained (Stilwell, J.L.,and Wyrobek, A.J. Proceedings of the Genetics Society annual meeting, San Francisco, July 1990) .
  • Two clones were identified and sequenced, and ⁇ hown to have 446 base pairs, including an open reading frame encoding a protein corresponding precisely to the known amino acid sequence of human PI. These clones were termed pBhpl-1.
  • the human PI gene cDNA, pBhpl-1 was digested with PstI and Ncol and treated with alkaline phosphatase. The fragment from the start codon to the 3' end was ligated to mouse promoter sequences derived from the mouse protamine PI and P2 gene ⁇ via an Ncol re ⁇ triction ⁇ ite at the start codon.
  • Various combination ⁇ of the human PI gene, mouse PI or P2 promoter, 5' and 3' UTRs and terminators were used in the constructs. The composition of the various constructs are shown in Table 4 and Figure 1.
  • mouse protamine-1 prm-l
  • protamine-2 prm-l
  • human protamine-1 HI gene.
  • human PI HI
  • P2 H2
  • Two primers were constructed to amplify (by the polymera ⁇ e chain reaction, PCR) both endogenou ⁇ mouse protamine, and the transgene construct.
  • One primer recognized the 5' end of the mouse gene and therefore hybridized to both the endogenou ⁇ gene and the tran ⁇ gene, while the other two recognized sequences 533bp and 308bp downstream of the first primer in the mouse gene and the tran ⁇ gene, respectively. Therefore, transgenic mice are identifiable by the presence of both bands, whereas non-transgenic mice only have the band representing the endogenous gene.
  • Offspring were tested for transgene incorporation using a 1 cm section of tail that was obtained several weeks after birth. Tis ⁇ ue samples were digested with proteinase K at 50-55°C overnight.
  • This lysate was extracted twice with phenol/chloroform and the DNA precipitated with ethanol and amplified by PCR using the primers de ⁇ cribed above. Off ⁇ pring were rai ⁇ ed until they were fertile (8-10 week ⁇ ) and were then mated to produce at least 10 male progeny each. Primers were synthesized for the specific detection of the recombinant molecule.
  • Transgenic mice were produced by the injection of male pronuclei with recombinant DNA prepared as above. Each experiment involved two consecutive days of injections. Each day involved the following: (1) isolating 100 to 160 fertilized eggs from 6 superovulated female mice; (2) injecting 60 to 90 pronuclear eggs with recombinant DNA; and (3) transferring 30 eggs to each of two recipient female mice.
  • the human Pi gene was recombined with the mouse P2 promoter sequence ( Figure l, Construct 1) and the recombinant construct injected into pronuclei of fertilized mouse eggs. This con ⁇ truct proved to be lethal to the embryo. In the litter ⁇ acrificed 10 days after conception, two embryos that carried the transgene were small and withered in appearance compared with two nontransgenic litter mates. A second recombinant protamine construct
  • Transgenic mice sperm were analyzed for the presence of human PI as follows.
  • Mouse sperm were collected using conventional techniques.sperm nuclei were i ⁇ olated free of tail, acro ⁇ omal and cytopla ⁇ ic co ponent ⁇ a ⁇ de ⁇ cribed in Example 1 (Balhorn, R. , et al., Biochemistry (1977) J 6:4074) . Nuclei were prepared using mixed alkyl- trimethylammonium bromide and DTT (MATAB/DTT) .
  • the nuclei were labeled with Hoechst 33258 dye (Somerville, NJ) to mark the DNA and were ⁇ creened for PI and P2a+P2b by reacting with a fluore ⁇ cein-tagged monoclonal antibody specific for human PI (hup IM) and P2a+P2b (hup 2b) , produced as described in Example 1, under conditions that maintained binding specificitie ⁇ .
  • the antibodies were also reacted with human sperm nuclei, prepared as described in Example 1, as a control. Binding of monoclonal antibodies to sperm nuclei wa ⁇ mea ⁇ ured by dual beam flow cytometry and modal labeling intensities increased with both increased antibody concentration and increased duration of staining.
  • Human and mouse sperm nuclei were identified by their different scatter signals when unstained samples of the respective populations were analyzed on flow cytometry graphs. The specificity of the antibodies was demonstrated by mixing prepared human sperm nuclei with similarly prepared mouse ⁇ perm nuclei in the presence of protamine-specific antibodies, hup IM and hup la. Protamine-specific binding activity was correlated with other size and shape parameters, as determined by image analysis, which verify the presence of sperm at the position ⁇ evaluated.
  • the designated deposit ⁇ will be maintained for a period of thirty (30) years from the date of deposit, or for five (5) years after the last request for the deposit; or for the enforceable life of the U.S. patent, whichever is longer. Should a culture become nonviable or be inadvertently destroyed, or, in the case of plasmid-containing strains, lose its plasmid, it will be replaced with a viable culture(s) of the same taxonomic description.

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Abstract

L'invention concerne des procédés d'identification de mutagènes germinaux, ainsi que des produits de recombinaison de transgènes et des animaux transgéniques s'utilisant dans lesdits procédés. On peut identifier des agents mutagènes au moyen du sperme provenant de l'animal transgénique qui comprendra un gène codant une protéine hétérologue de cellule de sperme, un gène codant un mutant de la protéine de cellule de sperme, ou un gène variant incapable de transcription sauf en cas d'apparition d'une mutation. Si l'animal est exposé à un agent germinal mutagène, des mutations apparaîtront dans le gène de la protéine de cellule de sperme, modifiant ainsi la spécificité du produit génique et permettant de détecter ledit produit.
PCT/US1994/009724 1993-09-10 1994-08-25 Procede d'identification d'agents mutagenes au moyen de sperme de souris transgeniques WO1995007106A1 (fr)

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Publication number Priority date Publication date Assignee Title
US6462170B1 (en) * 1997-03-20 2002-10-08 Fondazione Centro San Raffaele Del Monte Tabor UPAR mimicking peptide

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AMERICAN JOURNAL OF HUMAN GENETICS, Volume 37, Number 4 Supplement, issued 1985, A.J. WYROBEK et al., "Monoclonal Antibodies to Human Sperm Protamines", page A22. *
ANNALS OF THE NEW YORK ACADEMY OF SCIENCES, Volume 564, issued 1989, J.J. PESCHON et al., "Expression of Mouse Protamine 1 Genes in Transgenic Mice", pages 186-197. *
ENVIRONMENTAL MOLECULAR MUTAGENESIS, Volume 17, Number Supplement 1, issued 1991, A.J. WYROBEK et al., "Cytometric Detection and Analysis of Rare Human Sperm Nuclei That Don't Bind Protamine-specific Monoclonal Antibodies", page 78. *
GENOMICS, Volume 8, issued 1990, L. DOMENJOUD et al., "Genomic Sequences of Human Protamines Whose Genes, PRM1 and PRM2, are Clustered", pages 127-133. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 77, Number 12, issued December 1980, A.A. ANSARI et al., "In Vivo Germinal Mutation Detection With 'Monospecific' Antibody Against Lactate Dehydrogenase-X", pages 7352-7356. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6462170B1 (en) * 1997-03-20 2002-10-08 Fondazione Centro San Raffaele Del Monte Tabor UPAR mimicking peptide

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