EP1130960A4 - Transgene tiere - Google Patents

Transgene tiere

Info

Publication number
EP1130960A4
EP1130960A4 EP99960310A EP99960310A EP1130960A4 EP 1130960 A4 EP1130960 A4 EP 1130960A4 EP 99960310 A EP99960310 A EP 99960310A EP 99960310 A EP99960310 A EP 99960310A EP 1130960 A4 EP1130960 A4 EP 1130960A4
Authority
EP
European Patent Office
Prior art keywords
protein
genome
virus
leu
polynucleotide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99960310A
Other languages
English (en)
French (fr)
Other versions
EP1130960A1 (de
Inventor
Robert D Bremel
Gregory T Bleck
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wisconsin Alumni Research Foundation
Original Assignee
Wisconsin Alumni Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wisconsin Alumni Research Foundation filed Critical Wisconsin Alumni Research Foundation
Publication of EP1130960A1 publication Critical patent/EP1130960A1/de
Publication of EP1130960A4 publication Critical patent/EP1130960A4/de
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • 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
    • A01K2227/101—Bovine
    • 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
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • 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
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to improved methods for the generation of transgenic non-human animals.
  • the present invention relates to the introduction of retroviral particles into the perivitelline space of gametes, zygotes and early stage embryos to allow the insertion of genetic material into the genome of the recipient gamete or embryo.
  • BACKGROUND The ability to alter the genetic make-up of animals, such as domesticated mammals such as cows, pigs and sheep, allows a number of commercial applications. These applications include the production of animals which express large quantities of exogenous proteins in an easily harvested form (e.g., expression into the milk), the production of animals which are resistant to infection by specific microorganisms and the production of animals having enhanced growth rates or reproductive performance. Animals which contain exogenous DNA sequences in their genome are referred to as transgenic animals.
  • the most widely used method for the production of transgenic animals is the microinjection of DNA into the pronuclei of fertilized embryos. This method is efficient for the production of transgenic mice but is much less efficient for the production of transgenic animals using large mammals such as cows and sheep. For example, it has been reported that 1,000 to 2,000 bovine embryos at the pronuclear stage must be microinjected to produce a single transgenic cow at an estimated cost of more than $500,000 (Wall et al, J. Cell. Biochem. 49:113 [1992]). Furthermore, microinjection of pronuclei is more difficult when embryos from domestic livestock (e.g., cattle, sheep, pigs) is employed as the pronuclei are often obscured by yolk material. While techniques for the visualization of the pronuclei are known (i.e., centrifugation of the embryo to sediment the yolk), the injection of pronuclei is an invasive technique which requires a high degree of operator skill.
  • transgenic animals are typically mosaic for the transgene since incorporation occurs only in a subset of cells which form the transgenic animal.
  • the consequences of mosaic incorporation of retroviral sequences include lack of transmission of the transgene to progeny due to failure of the retrovirus to integrate into the germ line, difficulty in detecting the presence of viral sequences in the founder mice in those cases where the infected cell contributes to only a small part of the fetus and difficulty in assessing the effect of the genes carried on the retrovirus.
  • transgenic sheep The efficiency of producing transgenic sheep was low (efficiency is defined as the number of transgenics produced compared to the number of embryos manipulated); only 4.2% of the injected embryos were found to be transgenic.
  • Haskell and Bowen describe the micro-injection of mitomycin C-treated cells producing retrovirus into the perivitelline space of 1 to 4 cell bovine embryos.
  • the use of virus-producing cells precludes the delivery of a controlled amount of viral particles per embryo.
  • the resulting fetuses contained between 2 and 12 proviruses and were shown to be mosaic for proviral integration sites, the presence of provirus, or both.
  • the efficiency of producing transgenic bovine embryos was low, only 7% of the injected embryos were found to be transgenic.
  • the art needs improved methods for the production of transgenic animals, particularly for the production of transgenics using large domestic livestock animals.
  • the ideal method would be simple to perform and less invasive than pronuclear injection, efficient, would produce mosaic transgenic founder animals at a low frequency and would result in the integration of a defined number of copies of the introduced sequences into the genome of the transgenic animal.
  • the present invention provides improved methods and compositions for the production of transgenic non-human animals.
  • the present invention provides a composition comprising a non-human unfertilized oocyte comprising a heterologous ohgonucleotide (i.e., a heterologous polynucleotide) integrated into the genome of the oocyte.
  • a heterologous ohgonucleotide i.e., a heterologous polynucleotide
  • the unfertilized oocyte is a pre-maturation oocyte.
  • the unfertilized oocyte is a pre-fertilization oocyte.
  • the present invention is not limited by the nature of the heterologous ohgonucleotide contained within the genome of the oocyte.
  • the heterologous ohgonucleotide is the proviral form of a retroviral vector.
  • Retroviral vectors containing a variety of genes may be employed.
  • the retroviral vector may contain sequences encoding proteins which modify growth rate, size and/or carcass composition (e.g., bovine growth hormone or other growth hormones) or foreign proteins of commercial value that are expressed in, and harvested from, a particular tissue component (e.g., blood or milk).
  • the retroviral vector may contain genes that confer disease resistance to viruses or other microorganisms, including DNA sequences that are transcribed into RNA sequences that catalytically cleave specific RNAs (Le., ribozymes) such as viral RNAs and DNA sequences that are transcribed into anti-sense RNA of an essential gene of a pathogenic microorganism.
  • RNA sequences that catalytically cleave specific RNAs (Le., ribozymes)
  • viral RNAs and DNA sequences that are transcribed into anti-sense RNA of an essential gene of a pathogenic microorganism.
  • the above protein-encoding genes and DNA sequences are examples of "genes of interest"
  • compositions of the present invention are not limited by the nature of the non- human animal employed to provide oocytes.
  • the non-human animal is a mammal (e.g., cows, pigs, sheep, goats, rabbits, rats, mice, etc.).
  • the non-human animal is a cow.
  • the present invention further provides a method for introducing a heterologous polynucleotide into the genome of a non-human unfertilized oocyte, comprising: a) providing: i) a non-human unfertilized egg comprising an oocyte having a plasma membrane and a zona pellucida, the plasma membrane and the zona pellucida defining a perivitelline space; ii) an aqueous solution comprising a heterologous polynucleotide; and b) introducing the solution comprising the heterologous polynucleotide into the perivitelline space under conditions which permit the introduction of the heterologous polynucleotide into the genome of the oocyte.
  • the method of the present invention is not limited by the nature of the heterologous polynucleotide employed.
  • the heterologous polynucleotide encodes a protein of interest.
  • the heterologous polynucleotide is contained within genome of a recombinant retrovirus.
  • the method of the present invention may be practiced using unfertilized eggs comprising a pre-maturation oocyte. Alternatively, the method of the present invention may employ pre-fertilization oocytes as the unfertilized egg.
  • infectious retroviral particles comprising the heterologous polynucleotide are preferentially employed.
  • the method of the present invention is not limited by the nature of the infectious retrovirus employed to deliver nucleic acid sequences to an oocyte. Any retrovirus which is capable of infecting the species of oocyte to be injected may be employed.
  • the infectious retrovirus comprises a heterologous membrane-associated protein.
  • the heterologous membrane-associated protein is a G glycoprotein selected from a virus within the family Rhabdoviridae.
  • the heterologous membrane-associated protein is selected from the group consisting of the G glycoprotein of vesicular stomatitis virus, Piry virus, Chandipura virus, Spring viremia of carp virus and Mokola virus.
  • the heterologous membrane-associated protein is the G glycoprotein of vesicular stomatitis virus.
  • the method of the present invention is not limited by the nature of the non-human animal employed to provide oocytes.
  • the non-human animal is a mammal (e.g., cows, pigs, sheep, goats, rabbits, rats, mice, etc.).
  • the non-human animal is a cow.
  • the present invention further provides a method for the production of a transgenic non-human animal comprising: a) providing: i) an unfertilized egg comprising an oocyte having a plasma membrane and a zona pellucida, the plasma membrane and the zona pellucida defining a perivitelline space; ii) an aqueous solution containing infectious retrovirus; b) introducing the solution containing infectious retrovirus into the perivitelline space under conditions which permit the infection of the oocyte; and c) contacting the infected oocyte with sperm under conditions which permit the fertilization of the infected oocyte to produce an embryo.
  • the method of the present invention further comprises, following the fertilization of the infected oocyte, the step of transferring the embryo into a hormonally synchronized non-human recipient animal (i.e., a female animal hormonally synchronized to stimulate early pregnancy).
  • the method comprises the step of allowing the transferred embryo to develop to term.
  • at least one transgenic offspring is identified from the offspring allowed to develop to term.
  • the method of the present invention may be practiced using unfertilized eggs comprising a pre-maturation oocyte. Alteratively, the method of the present invention may employ pre-fertilization oocytes as the unfertilized egg.
  • the method may further comprise, following the introduction of the solution containing infectious retrovirus into the pre-maturation oocyte, the further step of culturing the infected pre- maturation oocyte under conditions which permit the maturation of the pre-maturation oocyte.
  • the art is well aware of culture conditions which permit the in vitro maturation of pre- maturation oocytes from a variety of mammalian species.
  • the method of the present invention is not limited by the nature of the infectious retrovirus employed to deliver nucleic acid sequences to an oocyte. Any retrovirus which is capable of infecting the species of oocyte to be injected may be employed.
  • the infectious retrovirus comprises a heterologous membrane-associated protein.
  • the heterologous membrane-associated protein is a G glycoprotein selected from a virus within the family Rhabdoviridae.
  • the heterologous membrane-associated protein is selected from the group consisting of the G glycoprotein of vesicular stomatitis virus, Piry virus, Chandipura virus, Spring viremia of carp virus and Mokola virus.
  • the heterologous membrane- associated protein is the G glycoprotein of vesicular stomatitis virus.
  • the method of the present invention is not limited by the nature of the non-human animal employed to provide oocytes.
  • the non-human animal is a mammal (e.g., cows, pigs, sheep, goats, rabbits, rats, mice, etc.).
  • the non-human animal is a bovine.
  • the present invention further provides compositions comprising a stably maintained recombinant mammalian zygote, wherein the zygote comprises a polynucleotide containing th proviral form of a retroviral vector integrated into the genome of the zygote.
  • the mammalian zygote is a bovine zygote, while in other preferred embodiments, the zygote is any mammalian zygote. Indeed, it is not intended that the present invention be limited to any particular animal species.
  • the proviral form of the retroviral vector encodes a protein of interest.
  • the recombinant retroviral vector comprises Moloney murine leukemia virus LTR. However, it is not intended that the present invention be limited to any particular retroviral LTR. Indeed, it is contemplated that other retroviral LTRs, including, but not limited, to mouse mammary tumor virus LTR, will find use in the present invention.
  • the present invention also provides methods for introducing a polynucleotide contained within the genome of a recombinant retrovirus into the genome of a mammalian zygote, comprising: a) providing: i) a mammalian zygote having a plasma membrane and a zona pellucida, wherein the plasma membrane and zona pellucida define a perivitelline space; ii) an aqueous solution comprising a polynucleotide contained within the genome of a recombinant retrovirus; and b) introducing the solution comprising the polynucleotide contained within the genome of a recombinant retrovirus into the perivitelline space, under conditions which permit the introduction of the polynucleotide contained within the genome of the recombinant retrovirus into the genome of the zygote, such that the polynucleotide is stably maintained.
  • the efficiency of the introduction of the polynucleotide into the genome of the zygote is at least twenty percent. I still other embodiments, the efficiency ranges from approximately twenty percent to one hundred percent.
  • the polynucleotide contained within th genome of the recombinant retrovirus encodes a protein of interest.
  • the method further comprises the step of transferring the zygote into a mammalian female recipient that is hormonally synchronized to simulate early pregnancy, thereby giving a transferred embryo.
  • the method further comprises the step of allowing the transferred embryo to develop to term.
  • the method comprises the additional step of identifying at least one transgenic offspring.
  • the present invention provides transgenic animals produced according to the above methods.
  • the mammalian zygote is a bovine zygote, while in other preferred embodiments, the zygote is any other mammalian zygote. Indeed, is not intended that the present invention be limited to any particular animal species.
  • the recombinant retrovirus comprises Moloney murine leukemia virus long terminal repeat.
  • the present invention be limited to any particular retroviral LTR. Indeed, it is contemplated that other retroviral LTRs, including, but not limited to mouse mammary tumor virus LTR, will find use in the present invention.
  • the protein of interest is expressed by the transgenic offspring. In some embodiments, the protein of interest is expressed in at least one body fluid of the transgenic offspring. In some particularly preferred embodiments, the expression of the protein of interest is preferentially mammary-specific expression.
  • the recombinant retrovirus comprises a heterologous membrane-associated protein.
  • the heterologous membrane-associated protein is a G glycoprotein selected from a virus within the family Rhabdoviridae.
  • the G glycoprotein is selected from the group comprising the G glycoprotein of vesicular stomatitis virus, Piry virus, Chandipura virus, Spring viremia of carp virus, Rabies virus, and okola virus.
  • the present invention also provides methods for producing transgenic non-human animals, wherein the genome of the transgenic non-human animal comprises a polynucleotide encoding a recombinant retrovirus and at least one protein of interest, comprising the steps of: a) providing: i) a non-human mammalian zygote having a plasma membrane and a zona pellucida, wherein the plasma membrane and the zona pellucida define a perivitelline space; ii) an aqueous solution comprising a polynucleotide contained within the genome of a recombinant retrovirus; b) introducing the solution comprising the polynucleotide contained within the genome of a recombinant retrovirus into the perivitelline space under conditions which permit the introduction of the polynucleotide contained within the genome of a recombinant retrovirus into the genome of the zygote, such that the polynucleotide is stably maintained in a re
  • the recombinant retrovirus comprises Moloney murine leukemia virus long terminal repeat.
  • the efficiency of the introduction of the polynucleotide is at least twenty percent In still other embodiments, the efficiency ranges from approximately twenty percent to one hundred percent.
  • the expression of the polynucleotide is preferentially mammary-specific expression.
  • the methods comprise the further step of mating the transgenic animal to a non-transgenic animal under conditions such that transgenic offspring are produced.
  • the transgenic offspring express the polynucleotide.
  • the expression of the polynucleotide is mammary-specific expression.
  • the mammalian zygote is a bovine zygote, while in other preferred embodiments, the zygote is any other mammalian zygote. Indeed, is not intended that the present invention be limited to any particular animal species.
  • the present invention also provides methods for expressing a protein of interest, wherein the protein of interest is encoded by a polynucleotide contained within the genome of a recombinant retrovirus, comprising the steps of: a) providing: i) a non-human mammalian zygote having a plasma membrane and a zona pellucida, wherein the plasma membrane and the zona pellucida define a perivitelline space; ii) an aqueous solution comprising a polynucleotide encoding a protein of interest contained within the genome of a recombinant retrovirus; and b) introducing the solution comprising the polynucleotide encoding a protein of interest contained within the genome of a recombinant retrovirus into the perivitelline space, under conditions which permit the introduction of the polynucleotide contained within the genome of a recombinant retrovirus into the genome of the zygote, such that the polynucleotide
  • the recombinant retrovirus comprises Moloney murine leukemia virus long terminal repeat.
  • the present invention be limited to any particular retroviral LTR. Indeed, it is contemplated that other retroviral LTRs, including, but not limited, to mouse mammary tumor virus LTR, will find use in the present invention.
  • introduction of the polynucleotide into the genome of the zygote is at least twenty percent. In still other embodiments, the efficiency ranges from approximately twenty percent to one hundred percent.
  • the polynucleotide contained within the genome of a recombinant retrovirus encodes a viral protein.
  • viral protein is hepatitis B surface antigen.
  • the present invention provides protein produced according to the above methods.
  • the method further comprises the step of harvesting the expressed protein of interest
  • the expressed protein is expressed in the body fluids of the non-human animal.
  • body fluids are selected from the group consisting of blood, milk, semen, and urine.
  • the mammalian zygote is a bovine zygote, while in other preferred embodiments, the zygote is any mammalian zygote. Indeed, it is not intended that the present invention be limited to any particular animal species.
  • the present invention also provides methods for expressing a protein of interest wherein the protein of interest is encoded by a polynucleotide contained within the genome of a recombinant retrovirus, and the polynucleotide is integrated into the genome of a mammalian unfertilized oocyte, comprising the steps of: a) providing: i) an unfertilized mammalian egg comprising an oocyte having a plasma membrane and a zona pellucida, wherein the plasma membrane and the zona pellucida define a perivitelline space; ii) an aqueous solution containing recombinant retrovirus, wherein the recombinant retrovirus comprises a polynucleotide encoding a protein of interest; b) introducing the solution containing recombinant retrovirus into the perivitelline space under conditions which permit the infection of the oocyte to provide an infected oocyte; c) contacting the infected oocyte with sper
  • the unfertilized oocyte is a pre-maturation oocyte.
  • the method comprises the further step of culturing the infected pre-maturation oocyte under conditions which permit the maturation of the pre-maturation oocyte.
  • the unfertilized oocyte is a pre-fertilization oocyte.
  • the method further comprises the step of identifying at least one transgenic offspring.
  • the mammal is a bovine.
  • the present invention be limited to any particular animal species.
  • the recombinant retrovirus comprises Moloney murine leukemia virus long terminal repeat
  • the present invention be limited to any particular retroviral LTR. Indeed, it is contemplated that other retroviral LTRs, including, but not limited, to mouse mammary tumor virus LTR, will find use in the present invention.
  • the expression of the protein of interest is preferentially mammary specific expression.
  • the introduction of the polynucleotide into the genome of the infected oocyte is greater than twenty percent. In still other embodiments, the efficiency ranges from approximately twenty percent to one hundred percent.
  • the polynucleotide contained within the genome of a recombinant retrovirus encodes a viral protein.
  • the viral protein is hepatitis B surface antigen.
  • the expressed protein is expressed in the body fluids of the mammalian animal.
  • the body fluids are selected from the group consisting of blood, milk, semen, and urine.
  • the methods further comprise the step of f) harvesting the expressed protein of interest.
  • the present invention also provides a protein of interest expressed using the above methods.
  • the recombinant retrovirus comprises a heterologous membrane-associated protein.
  • the heterologous membrane-associated protein is a G glycoprotein selected from a virus within the family Rhabdoviridae.
  • the G glycoprotein is selected from the group comprising the G glycoprotein of vesicular stomatitis virus, Piry virus, Chandipura virus, Spring viremia of carp virus and Mokola virus.
  • Figure 1 provides a schematic showing the production of pre-maturation oocytes, pre- fertilization oocytes and fertilized oocytes (zygotes).
  • Figure 2 shows an autoradiogram of a Southern blot of genomic DNA isolated from the skin (A) and blood (B) of calves derived from pre-fertilization oocytes and zygotes which were injected with pseudotyped LSRNL retrovirus.
  • Figure 3 shows an ethidium bromide stained agarose gel containing electrophoresed PCR products which were amplified using neo gene primers (A) or HBsAg primers (B) from the blood and skin of calves derived from pre-fertilization oocytes and zygotes injected with pseudotyped LSRNL retrovirus.
  • Figure 4 shows an ethidium bromide stained agarose gel containing electrophoresed
  • the term “egg,” when used in reference to a mammalian egg, means an oocyte surrounded by a zona pellucida and a mass of cumulus cells (follicle cells) with their associated proteoglycan.
  • the term “egg” is used in reference to eggs recovered from antral follicles in an ovary (these eggs comprise pre-maturation oocytes) as well as to eggs which have been released from an antral follicle (a ruptured follicle).
  • the term “oocyte” refers to a female gamete cell and includes primary oocytes, secondary oocytes and mature, unfertilized ovum.
  • An oocyte is a large cell having a large nucleus (i.e., the germinal vesicle) surrounded by ooplasm.
  • the ooplasm contains non- nuclear cytoplasmic contents including mRNA, ribosomes, mitochondria, yolk proteins, etc.
  • the membrane of the oocyte is referred to herein as the "plasma membrane.”
  • pre-maturation oocyte refers to a female gamete cell following the oogonia stage (i.e., mitotic proliferation has occurred) that is isolated from an ovary (e.g., by aspiration) but which has not been exposed to maturation medium in vitro.
  • ovary e.g., by aspiration
  • Those of skill in the art know that the process of aspiration causes oocytes to begin the maturation process but that completion of the maturation process (i.e., formation of a secondary oocyte which has extruded the first polar body) in vitro requires the exposure of the aspirated oocytes to maturation medium.
  • Pre-maturation oocytes will generally be arrested at the first anaphase of meiosis.
  • pre-fertilization oocyte refers to a female gamete cell such as a pre-maturation oocyte following exposure to maturation medium in vitro but prior to exposure to sperm (i.e., matured but not fertilized).
  • the pre-fertilization oocyte has completed the first meiotic division, has released the first polar body and lacks a nuclear membrane (the nuclear membrane will not reform until fertilization occurs; after fertilization, the second meiotic division occurs along with the extrusion of the second polar body and the formation of the male and female pronuclei).
  • Pre-fertilization oocytes may also be referred to as matured oocytes at metaphase II of the second meiosis.
  • unfertilized egg or “unfertilized oocyte” as used herein, refers to any female gamete cell which has not been fertilized and these terms encompass both pre- maturation and pre-fertilization oocytes.
  • zygote refers to a fertilized oocyte that has not yet undergone the first cleavage step in the development of an embryo (i.e., it is at the single-cell stage).
  • peripheral space refers to the space located between the zona pellucida and the plasma membrane of a mammalian egg or oocyte.
  • trans is used in reference to the positioning of genes of interest on the different strands of nucleic acid (e.g., alleles present on the two chromosomes of a chromosomal pair).
  • trans-acting is used in reference to the controlling effect of a regulatory gene on a gene present on a different chromosome. In contrast to promoters, repressors are not limited in their binding to the DNA molecule that includes their genetic information. Therefore, repressors are sometimes referred to as trans-acting control elements.
  • trans-activation refers to the activation of gene sequences by factors encoded by a regulatory gene which is not necessarily contiguous with the gene sequences which it binds to and activates.
  • cis is used in reference to the presence of genes on the same chromosome.
  • cis-acting is used in reference to the controlling effect of a regulatory gene on a gene present on the same chromosome. For example, promoters, which affect the synthesis of downstream mRNA are cis-acting control elements.
  • retrovirus is used in reference to RNA viruses which utilize reverse transcriptase during their replication cycle (i.e., retroviruses are incapable of replication; rather, these are useful RNA sequences that are packaged with at least two enzymes that are required for the insertion of the RNA sequences into the host cell genome).
  • the retroviral genomic RNA is converted into double-stranded DNA by reverse transcriptase.
  • provirus This double-stranded DNA form of the virus integrates into the chromosome of the infected cell and is referred to as a "provirus.”
  • proviral is used in reference to constructs that are similar to "retrotransposons.” These are integrated genes that are bracketed by LTRs in the host cell genome. However, in preferred embodiments, the proviral constructs cannot replicate.
  • the provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules which encode the structural proteins and enzymes needed to produce new viral particles. At each end of the provirus are structures called “long terminal repeats" or "LTRs".
  • the LTR contains numerous regulatory signals including transcriptional control elements, polyadenylation signals and sequences needed for replication and integration of the viral genome.
  • the viral LTR is divided into three regions called U3, R and U5.
  • the U3 region contains the enhancer and promoter elements.
  • the U5 region contains the polyadenylation signals.
  • the R (repeat) region separates the U3 and U5 regions and transcribed sequences of the R region appear at both the 5' and 3' ends of the viral RNA
  • provirus is used in reference to a virus that is integrated into a host cell chromosome (or genome), and is transmitted from one cell generation to the next without causing lysis or destruction of the host cell.
  • the term is also used in reference to a duplex DNA sequence present in an eukaryotic chromosome, which corresponds to the genome of an RNA retrovirus.
  • endogenous virus is used in reference to an inactive virus which is integrated into the chromosome of its host cell (often in multiple copies), and can thereby exhibit vertical transmission. Endogenous viruses can spontaneously express themselves and may result in malignancies.
  • amphotrope and “amphotropic” are used in reference to endogenous viruses that readily multiply in cells of the species in which they were induced, as well as cells of other species.
  • ecotrope and "ecotropic” are used in reference to endogenous viruses that multiply readily in cells of the species in which they were induced, but cannot multiply in cells of other species.
  • xenotrope and "xenotropic” are used in reference to endogenous viruses that cannot infect cells of the species in which they were induced, but can infect and multiply in cells of other species.
  • infectious retrovirus refers to a retroviral particle which is capable of entering a cell (i.e., the particle contains a membrane-associated protein such as an envelope protein or a viral G glycoprotein which can bind to the host cell surface and facilitate entry of the viral particle into the cytoplasm of the host cell) and integrating the retroviral genome (as a double-stranded provirus) into the genome of the host cell.
  • a membrane-associated protein such as an envelope protein or a viral G glycoprotein which can bind to the host cell surface and facilitate entry of the viral particle into the cytoplasm of the host cell
  • retroviral genome as a double-stranded provirus
  • retroviral vector is used in reference to retroviruses which have been modified so as to serve as vectors for introduction of nucleic acid into cells.
  • vector is used in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another. Retroviral vectors transfer RNA, which is then reverse transcribed into DNA.
  • vehicle is sometimes used interchangeably with “vector.”
  • expression vector refers to a recombinant molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism.
  • Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences.
  • Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
  • operable combination refers to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced.
  • the term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
  • the term "protein of interest” refers to any protein for which expression is desired.
  • the term encompasses any recombinant forms of a protein that is desired.
  • the term "gene of interest” refers to any gene that is desired.
  • the gene of interest encodes at least a portion of a protein of interest.
  • the term "genetic cassette” as used herein refers to a fragment or segment of nucleic acid containing a particular grouping of genetic elements. The cassette can be removed and inserted into a vector or plasmid as a single unit.
  • LTR long terminal repeat
  • retroviral vectors can be used to transfer genes efficiently into host cells by exploiting the viral infectious process.
  • Foreign or heterologous genes cloned (j ' .e., inserted using molecular biological techniques) into the retroviral genome can be delivered efficiently to host cells which are susceptible to infection by the retrovirus.
  • the replicative capacity of the retroviral genome can be destroyed.
  • the resulting replication-defective vectors can be used to introduce new genetic material to a cell but they are unable to replicate.
  • a helper virus or packaging cell line can be used to permit vector particle assembly and egress from the cell.
  • vector particle refers to viral-like particles that are capable of introducing nucleic acid into a cell through a viral-like entry mechanism.
  • the host range of a retroviral vector i.e., the range of cells that these vectors can infect
  • packetaging signal or “packaging sequence” refers to non- coding sequences located within the retroviral genome which are required for insertion of the viral RNA into the viral capsid or particle.
  • Several retroviral vectors use the minimal packaging signal (also referred to as the psi sequence) needed for encapsidation of the viral genome. This minimal packaging signal encompasses bases 212 to 563 of the Mo-MuLV genome (Mann et al, Cell 33:153 [1983]).
  • extended packaging signal or extended packaging sequence refers to the use of sequences around the psi sequence with further extension into the gag gene.
  • this extended packaging sequence corresponds to the region encompassing base 1039 to base 1906 (Akagi et al, Gene 106:255 [1991]).
  • the frequently used M-MuLV vector, pLNL6 (Bender et al., J. Virol., 61:1639 [1987]), contains the entire 5' region of the genome including an extended packaging signal from bases 206 to 1039 of the Moloney murine sarcoma virus genome (numbering from Supplements and Appendices in RNA Tumor Viruses, 2nd Ed.
  • packaging cell lines is used in reference to cell lines that express viral structural proteins (e.g., gag, pol and env), but do not contain a packaging signal.
  • retroviral vector DNA When retroviral vector DNA is transfected into the cells, it becomes integrated into the chromosomal DNA and is transcribed, thereby producing full-length retroviral vector RNA that has a psi + sequence. Under these conditions, only the vector RNA is packaged into the viral capsid structures These complete, yet replication-defective, virus particles can then be used to deliver the retroviral vector to target cells with relatively high efficiency.
  • transfection refers to the introduction of foreign DNA into eukaryotic cells.
  • Transfection may be accomplished by a variety of means known in the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.
  • transduction refers to the delivery of a gene(s) using a retroviral vector by means of infection rather than by transfection.
  • membrane-associated protein refers to a protein (e.g., a viral envelope glycoprotein or the G proteins of viruses in the Rhabdoviridae family such as VSV, Piry, Chandipura and Mokola) which are associated with the membrane surrounding a viral particle; these membrane-associated proteins mediate the entry of the viral particle into the host cell.
  • the membrane associated protein may bind to specific cell surface protein receptors, as is the case for retroviral envelope proteins or the membrane-associated protein may interact with a phospholipid component of the plasma membrane of the host cell, as is the case for the G proteins derived from members of the Rhabdoviridae family.
  • heterologous membrane-associated protein refers to a membrane-associated protein which is derived from a virus which is not a member of the same viral class or family as that from which the nucleocapsid protein of the vector particle is derived.
  • Virtual class or family refers to the taxonomic rank of class or family, as assigned by the International Committee on Taxonomy of Viruses.
  • the term "Rhabdoviridae” refers to a family of enveloped RNA viruses that infect animals, including humans, and plants.
  • the Rhabdoviridae family encompasses the genus Vesiculovirus which includes vesicular stomatitis virus (VSV), Cocal virus, Piry virus, Chandipura virus, and Spring viremia of carp virus (sequences encoding the Spring viremia of carp virus are available under GenBank accession number U18101).
  • the G proteins of viruses in the Vesiculovirus genera are virally-encoded integral membrane proteins that form externally projecting homotrimeric spike glycoproteins complexes that are required for receptor binding and membrane fusion.
  • the G proteins of viruses in the Vesiculovirus genera have a covalently bound palmitic acid (C 16 ) moiety.
  • the amino acid sequences of the G proteins from the Vesiculoviruses are fairly well conserved.
  • the Piry virus G protein share about 38% identity and about 55% similarity with the VSV G proteins (several strains of VSV are known, e.g., Indiana, New Jersey, Orsay, San Juan, etc., and their G proteins are highly homologous).
  • the Chandipura virus G protein and the VSV G proteins share about 37% identity and 52% similarity.
  • the G proteins from non-VSV Vesiculoviruses may be used in place of the VSV G protein for the pseudotyping of viral particles.
  • the G proteins of the Lyssa viruses also share a fair degree of conservation with the VSV G proteins and function in a similar manner (e.g., mediate fusion of membranes) and therefore may be used in place of the VSV G protein for the pseudotyping of viral particles.
  • the Lyssa viruses include the Mokola virus and the Rabies viruses (several strains of Rabies virus are known and their G proteins have been cloned and sequenced).
  • the Mokola virus G protein shares stretches of homology (particularly over the extracellular and transmembrane domains) with the VSV G proteins which show about 31% identity and 48% similarity with the VSV G proteins.
  • Preferred G proteins share at least 25% identity, preferably at least 30% identity and most preferably at least 35% identity with the VSV G proteins.
  • the VSV G protein from which New Jersey strain (the sequence of this G protein is provided in GenBank accession numbers M27165 and M21557) is employed as the reference VSV G protein.
  • condition which permit the maturation of a pre-maturation oocyte refers to conditions of in vitro cell culture which permit the maturation of a pre-maturation oocyte to a mature ovum (e.g., a pre-fertilization oocyte). These culture conditions permit and induce the events which are associated with maturation of the pre-maturation oocyte including stimulation of the first and second meiotic divisions.
  • the term "remedial gene” refers to a gene whose expression is desired in a cell to correct an error in cellular metabolism, to inactivate a pathogen or to kill a cancerous cell.
  • selectable marker refers to the use of a gene which encodes an enzymatic activity that confers resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed. Selectable markers may be "dominant”; a dominant selectable marker encodes an enzymatic activity which can be detected in any eukaryotic cell line.
  • dominant selectable markers examples include the bacterial aminoglycoside 3' phosphotransferase gene (also referred to as the neo gene) which confers resistance to the drug G418 in mammalian cells, the bacterial hygromycin G phosphotransferase (hyg) gene which confers resistance to the antibiotic hygromycin and the bacterial xanthine-guanine phosphoribosyl transferase gene (also referred to as the gpt gene) which confers the ability to grow in the presence of mycophenolic acid.
  • Other selectable markers are not dominant in that there use must be in conjunction with a cell line that lacks the relevant enzyme activity.
  • non-dominant selectable markers include the thymidine kinase (tk) gene which is used in conjunction with tk' cell lines, the CAD gene which is used in conjunction with CAD- deficient cells and the mammalian hypoxanthine-guanine phosphoribosyl transferase (hprt) gene which is used in conjunction with hprf cell lines.
  • tk thymidine kinase
  • CAD CAD- deficient cells
  • hprt mammalian hypoxanthine-guanine phosphoribosyl transferase
  • complementarity are used in reference to polynucleotides (i.e., a sequence of nucleo tides) related by the base-pairing rules. For example, for the sequence “A-G-T,” is complementary to the sequence “T-C-A.” Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.
  • hybridization is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the T m of the formed hybrid, and the G:C ratio within the nucleic acids.
  • T m is used in reference to the "melting temperature.”
  • the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
  • nucleic acid is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. With “high stringency” conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences. Thus, conditions of "weak” or “low” stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually less.
  • amplifiable nucleic acid is used in reference to nucleic acids which may be amplified by any amplification method. It is contemplated that "amplifiable nucleic acid” will usually comprise "sample template.”
  • sample template refers to nucleic acid originating from a sample which is analyzed for the presence of “target” (defined below).
  • background template is used in reference to nucleic acid other than sample template which may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.
  • the term "primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH).
  • the primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products.
  • the primer is an oligodeoxyribonucleotide.
  • the primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
  • probe refers to an oligonucleotide (te., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of hybridizing to another oligonucleotide of interest Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present invention will be labelled with any "reporter molecule,” so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems.
  • enzyme e.g., ELISA, as well as enzyme-based histochemical assays
  • the oligonucleotide of interest i.e., to be detected
  • a reporter molecule i.e., to be labelled
  • both the probe and oligonucleotide of interest will be labelled. It is not intended that the present invention be limited to any particular detection system or label.
  • target refers to the region of nucleic acid bounded by the primers used for polymerase chain reaction. Thus, the “target” is sought to be sorted out from other nucleic acid sequences.
  • a “segment” is defined as a region of nucleic acid within the target sequence.
  • PCR polymerase chain reaction
  • the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule.
  • the primers are extended with a polymerase so as to form a new pair of complementary strands.
  • the steps of denaturation, primer annealing and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one "cycle”; there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target sequence.
  • the length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter.
  • PCR polymerase chain reaction
  • PCR it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32 P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment).
  • any oligonucleotide sequence can be amplified with the appropriate set of primer molecules.
  • the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.
  • nested primers refers to primers that anneal to the target sequence in an area that is inside the annealing boundaries used to start PCR (Mullis, et al., Cold Spring Harbor Symposia, Vol. II, pp.263-273 [1986]). Because the nested primers anneal to the target inside the annealing boundaries of the starting primers, the predominant PCR-amplified product of the starting primers is necessarily a longer sequence, than that defined by the annealing boundaries of the nested primers. The PCR-amplified product of the nested primers is an amplified segment of the target sequence that cannot therefore, anneal with the starting primers. Advantages to the use of nested primers include the large degree of specificity, as well as the fact that a smaller sample portion may be used and yet obtain specific and efficient amplification.
  • amplification reagents refers to those reagents (deoxyribonucleoside triphosphates, buffer, etc.), needed for amplification except for primers, nucleic acid template and the amplification enzyme.
  • amplification reagents along with other reaction components are placed and contained in a reaction vessel (test tube, microwell, etc.).
  • restriction endonucleases and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
  • the term "recombinant DNA molecule” as used herein refers to a DNA molecule which is comprised of segments of DNA joined together by means of molecular biological techniques.
  • DNA molecules are said to have "5' ends” and "3' ends” because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotides referred to as the "5' end” if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the "3' end” if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring.
  • a nucleic acid sequence even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends.
  • discrete elements are referred to as being "upstream” or 5' of the "downstream” or 3' elements. This terminology reflects the fact that transcription proceeds in a 5' to 3' fashion along the DNA strand.
  • the promoter and enhancer elements which direct transcription of a linked gene are generally located 5' or upstream of the coding region However, enhancer elements can exert their effect even when located 3' of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.
  • an oligonucleotide having a nucleotide sequence encoding a gene means a DNA sequence comprising the coding region of a gene or in other words the DNA sequence which encodes a gene product.
  • the coding region may be present in either a cDNA or genomic DNA form.
  • Suitable control elements such as enhancers promoters, splice junctions, polyadenylation signals, etc.
  • the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.
  • transcription unit refers to the segment of DNA between the sites of initiation and te ⁇ nination of transcription and the regulatory elements necessary for the efficient initiation and termination.
  • a segment of DNA comprising an enhancer/promoter, a coding region and a termination and polyadenylation sequence comprises a transcription unit.
  • regulatory element refers to a genetic element which controls some aspect of the expression of nucleic acid sequences.
  • a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region.
  • Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc. (defined infra).
  • Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis et al, Science 236:1237 [1987]). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types (for review see Voss et al, Trends Biochem. Sci.,
  • the SV40 early gene enhancer is very active in a wide variety of cell types from many mammalian species and has been widely used for the expression of proteins in mammalian cells (Dijkema et al, EMBO J., 4:761 [1985]).
  • Two other examples of promoter/enhancer elements active in a broad range of mammalian cell types are those from the human elongation factor l ⁇ gene (Uetsuki et al, J. Biol.
  • promoter/enhancer denotes a segment of DNA which contains sequences capable of providing both promoter and enhancer functions (i.e., the functions provided by a promoter element and an enhancer element, see above for a discussion of these functions).
  • promoter/promoter may be "endogenous” or “exogenous” or “heterologous.”
  • An “endogenous” enhancer/promoter is one which is naturally linked with a given gene in the genome.
  • an “exogenous” or “heterologous” enhancer/promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer/promoter.
  • factor refers to a protein or group of proteins necessary for the transcription or replication of a DNA sequence.
  • SV40 T antigen is a replication factor which is necessary for the replication of DNA sequences containing the SV40 origin of replication.
  • Transcription factors are proteins which bind to regulatory elements such as promoters and enhancers and facilitate the initiation of transcription of a gene. Promoters and enhancers may bind to specific factors which increase the rate of activity from the promoter or enhancer.
  • viral promoter and enhancers may bind to factors encoded by the virus such that the viral promoter or enhancer is "activated” in the presence of the viral factor (in a virus infected cell or in a cell expressing the viral factor).
  • the level of activity in the presence of the factor i.e., activity "induced” by the factor
  • Different promoters may have different levels of basal activity in the same or different cell types. When two different promoters are compared in a given cell type in the absence of any inducing factors, if one promoter expresses at a higher level than the other it is said to have a higher basal activity.
  • the activity of a promoter and or enhancer is measured by detecting directly or indirectly the level of transcription from the elements).
  • Direct detection involves quantitating the level of the RNA transcripts produced from that promoter and/or enhancer.
  • Indirect detection involves quantitation of the level of a protein, often an enzyme, produced from RNA transcribed from the promoter and/or enhancer.
  • An commonly employed assay for promoter or enhancer activity utilizes the chloramphenicol acetyltransferase (CAT) gene.
  • CAT chloramphenicol acetyltransferase
  • the level of enzymatic activity is proportional to the amount of CAT RNA transcribed by the cell line.
  • This CAT assay therefore allows a comparison to be made of the relative strength of different promoters or enhancers in a given cell line.
  • a promoter is said to express at "high” or “low” levels in a cell line this refers to the level of activity relative to another promoter which is used as a reference or standard of promoter activity.
  • Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and acceptor site (See e.g., Sambrook, J. et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York [1989], pp. 16.7-16.8).
  • a commonly used splice donor and acceptor site is the splice junction from the 16S RNA of SV40.
  • Efficient expression of recombinant DNA sequences in eukaryotic cells requires expression of signals directing the efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal and are a few hundred nucleotides in length.
  • the term "poly A site” or "poly A sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly A tail are unstable and are rapidly degraded.
  • the poly A signal utilized in an expression vector may be "heterologous" or
  • endogenous poly A signal is one that is found naturally at the 3' end of the coding region of a given gene in the genome.
  • a heterologous poly A signal is one which is one which is isolated from one gene and placed 3' of another gene.
  • a commonly used heterologous poly A signal is the SV40 poly A signal.
  • the SV40 poly A signal is contained on a 237 bp Bam YOJBcl I restriction fragment and directs both termination and polyadenylation (Sambrook, J., supra, at 16.6-16.7).
  • Eukaryotic expression vectors may also contain "viral replicons "or "viral origins of replication.”
  • Viral replicons are viral DNA sequences which allow for the extrachromosomal replication of a vector in a host cell expressing the appropriate replication factors.
  • Vectors which contain either the SV40 or polyoma virus origin of replication replicate to high copy number (up to 10 4 copies/cell) in cells that express the appropriate viral antigen.
  • Vectors which contain the replicons from bovine papillomavirus or Epstein-Barr virus replicate extrachromosomally at low copy number (-100 copies/cell).
  • stable transfection or "stably transfected” refers to the introduction and integration of foreign DNA into the genome of the transfected cell.
  • stable transfectant refers to a cell which has stably integrated foreign DNA into the genomic DNA.
  • the term "stably maintained” refers to characteristics of recombinant (i.e., transgenic) animals that maintain at least one of their recombinant elements (i.e., the element that is desired) through multiple generations.
  • the term encompass the characteristics of transgenic animals that are capable of passing the transgene to their offspring, such that the offspring are capable of maintaining the expression and/or transcription of the transgene. It is not intended that the term be limited to any particular organism or any specific recombinant element.
  • transient transfection or “transiently transfected” refers to the introduction of foreign DNA into a cell where the foreign DNA fails to integrate into the genome of the transfected cell.
  • the foreign DNA persists in the nucleus of the transfected cell for several days. During this time the foreign DNA is subject to the regulatory controls that govern the expression of endogenous genes in the chromosomes.
  • transient transfectant refers to cells which have taken up foreign DNA but have failed to integrate this DNA.
  • gene of interest refers to the gene inserted into the polylinker of an expression vector. When the gene of interest encodes a gene which provides a therapeutic function, the gene of interest may be alternatively called a remedial gene.
  • nucleic acid molecule encoding As used herein, the terms “nucleic acid molecule encoding,” “DNA sequence encoding,” and “DNA encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.
  • the term “adoptive transfer” is used in reference to the transfer of one function to another cell or organism.
  • transfer of an immune function is made from one organism to another through the transfer of immunologically competent cells.
  • the present invention provides improved methods for the production of transgenic animals.
  • the methods of the present invention provide, for the first time, the production of transgenic animals by the introduction of exogenous DNA into pre-maturation oocytes and mature, unfertilized oocytes (i.e., pre-fertilization oocytes) using retroviral vectors which transduce dividing cells (e.g., vectors derived from murine leukemia virus [MLV]).
  • retroviral vectors which transduce dividing cells
  • the present invention provides methods and compositions for cytomegalovirus promoter-driven, as well as mouse mammary tumor LTR expression of various recombinant proteins.
  • the human cytomegalovirus (CMV) promoter has been developed for use in retroviral vectors for driving the expression of various recombinant proteins, and cell lines have been infected with these vectors, with resultant recombinant protein expression.
  • the mouse mammary tumor virus (MMTV) LTR has been previously shown to control expression of a recombinant protein in transgenic mice (Yom et al, Animal Biotech., 4:89-107 [1993]). In these mouse lines, expression was predominately observed in the mammary gland and milk, but low expression was also observed in the salivary gland, spleen, lung and kidney. The transgenic mice used in this experiment were produced using typical microinjection techniques.
  • the present invention provides methods and compositions for the use of MMTV LTR-driven expression which avoids the need for microinjection techniques.
  • the MMTV LTR has been developed for use in retroviral vectors for driving the expression of various recombinant proteins, and cell lines have been infected with these vectors, with resultant recombinant protein expression.
  • Retroviruses and Retroviral Vectors The following Description of the Invention is divided into the following sections: I. Retroviruses and Retroviral Vectors; ⁇ . Integration of Retroviral DNA; HI. Introduction of Retroviral Vectors Into Gametes Before the Last Meiotic Division; IV. Detection of the Retrovirus Following Injection Into Oocytes or Embryos; and V. Expression of Foreign Proteins in Transgenic Animals.
  • Retroviruses family Retroviridae are divided into three groups: the spumaviruses (e.g., human foamy virus); the lentiviruses (e.g., human immunodeficiency virus and sheep visna virus) and the oncoviruses (e.g., MLV, Rous sarcoma virus).
  • the spumaviruses e.g., human foamy virus
  • the lentiviruses e.g., human immunodeficiency virus and sheep visna virus
  • the oncoviruses e.g., MLV, Rous sarcoma virus.
  • Retroviruses are enveloped (i.e., surrounded by a host cell-derived lipid bilayer membrane) single-stranded RNA viruses which infect animal cells.
  • a retrovirus infects a cell, its RNA genome is converted into a double-stranded linear DNA form (i.e., it is reverse transcribed).
  • the DNA form of the virus is then integrated into the host cell genome as a provirus.
  • the provirus serves as a template for the production of additional viral genomes and viral mRNAs. Mature viral particles containing two copies of genomic RNA bud from the surface of the infected cell.
  • the viral particle comprises the genomic RNA, reverse transcriptase and other pol gene products inside the viral capsid (which contains the viral gag gene products) which is surrounded by a lipid bilayer membrane derived from the host cell containing the viral envelope glycoproteins (also referred to as membrane-associated proteins).
  • retroviral vectors The organization of the genomes of numerous retroviruses is well known in the art and this has allowed the adaptation of the retroviral genome to produce retroviral vectors.
  • the production of a recombinant retroviral vector carrying a gene of interest is typically achieved in two stages.
  • the gene of interest is inserted into a retroviral vector which contains the sequences necessary for the efficient expression of the gene of interest (including promoter and/or enhancer elements which may be provided by the viral long terminal repeats [LTRs] or by an internal promoter/enhancer and relevant splicing signals), sequences required for the efficient packaging of the viral RNA into infectious virions (e.g., the packaging signal [Psi], the tRNA primer binding site [-PBS], the 3' regulatory sequences required for reverse transcription [+PBS] and the viral LTRs).
  • the LTRs contain sequences required for the association of viral genomic RNA, reverse transcriptase and integrase functions, and sequences involved in directing the expression of the genomic RNA to be packaged in viral particles.
  • many recombinant retroviral vectors lack functional copies of the genes which are essential for viral replication (these essential genes are either deleted or disabled); the resulting virus is said to be replication defective.
  • the vector DNA is introduced into a packaging cell line.
  • Packaging cell lines provide viral proteins required in trans for the packaging of the viral genomic RNA into viral particles having the desired host range (i.e., the viral-encoded gag, pol and env proteins). The host range is controlled, in part, by the type of envelope gene product expressed on the surface of the viral particle.
  • Packaging cell lines may express ecotrophic, amphotropic or xenotropic envelope gene products.
  • the packaging cell line may lack sequences encoding a viral envelope (env) protein. In this case the packaging cell line will package the viral genome into particles which lack a membrane-associated protein (e.g., an env protein).
  • the packaging cell line containing the retroviral sequences is transfected with sequences encoding a membrane-associated protein (e.g., the G protein of vesicular stomatitis virus
  • VSV virus-associated protein expressed by the transfected packaging cell line; these viral particles which contain viral genomic RNA derived from one virus encapsidated by the envelope proteins of another virus are said to be pseudotyped virus particles.
  • Viral vectors including recombinant retroviral vectors, provide a more efficient means of transferring genes into cells as compared to other techniques such as calcium phosphate- DNA co-precipitation or DEAE-dextran-mediated transfection, electroporation or microinjection of nucleic acids.
  • nucleic acid is a receptor-mediated process (i.e., the virus binds to a specific receptor protein on the surface of the cell to be infected).
  • the virally transferred nucleic acid once inside a cell integrates in controlled manner in contrast to the integration of nucleic acids which are not virally transferred; nucleic acids transferred by other means such as calcium phosphate-DNA co-precipitation are subject to rearrangement and degradation.
  • the most commonly used recombinant retroviral vectors are derived from the amphotropic Moloney murine leukemia virus (MoMLV) (Miller and Baltimore, Mol. Cell. Biol., 6:2895 [1986]).
  • the MoMLV system has several advantages: 1) this specific retrovirus can infect many different cell types, 2) established packaging cell lines are available for the production of recombinant MoMLV viral particles and 3) the transferred genes are permanently integrated into the target cell chromosome.
  • the established MoMLV vector systems comprise a DNA vector containing a small portion of the retroviral sequence (the viral long terminal repeat or "LTR" and the packaging or "psi" signal) and a packaging cell line.
  • the gene to be transfe ⁇ ed is inserted into the DNA vector.
  • the viral sequences present on the DNA vector provide the signals necessary for the insertion or packaging of the vector RNA into the viral particle and for the expression of the inserted gene.
  • the packaging cell line provides the viral proteins required for particle assembly (Markowitz et al., J. Virol., 62:1120 [1988]).
  • VSV The low titer and inefficient infection of certain cell types by MoMLV-based vectors has been overcome by the use of pseudotyped retroviral vectors which contain the G protein of VSV as the membrane associated protein. Unlike retroviral envelope proteins which bind to a specific cell surface protein receptor to gain entry into a cell, the VSV G protein interacts with a phospholipid component of the plasma membrane (Mastromarino et al., J. Gen. Virol., 68:2359 [1977]). Because entry of VSV into a cell is not dependent upon the presence of specific protein receptors, VSV has an extremely broad host range.
  • VSV G protein Pseudotyped retroviral vectors bearing the VSV G protein have an altered host range characteristic of VSV (i.e., they can infect almost all species of vertebrate, invertebrate and insect cells). Importantly, VSV G- pseudotyped retroviral vectors can be concentrated 2000-fold or more by ultracentrifugation without significant loss of infectivity (Burns et al., Proc. Natl. Acad. Sci. USA 90:8033 [1993]).
  • the VSV G protein has also been used to pseudotype retroviral vectors based upon the human immunodeficiency virus (HIV) (Naldini et al, Science 272:263 [1996]).
  • HIV human immunodeficiency virus
  • the VSV G protein may be used to generate a variety of pseudotyped retroviral vectors and is not limited to vectors based on MoMLV.
  • the present invention is not limited to the use of the VSV G protein when a viral G protein is employed as the heterologous membrane-associated protein within a viral particle.
  • Chandipura viruses that are highly homologous to the VSV G protein and, like the VSV G protein, contain covalently linked palmitic acid (Brun et al, Intervirol., 38:274 [1995]; and Masters et al, Virol., 171:285 [1990]).
  • the G protein of the Piry and Chandipura viruses can be used in place of the VSV G protein for the pseudotyping of viral particles.
  • the VSV G proteins of viruses within the Lyssa virus genera such as Rabies and Mokola viruses show a high degree of conservation (amino acid sequence as well as functional conservation) with the VSV G proteins.
  • the Mokola virus G protein has been shown to function in a manner similar to the VSV G protein (i.e., to mediate membrane fusion) and therefore may be used in place of the VSV G protein for the pseudotyping of viral particles (Mebatsion et al., J. Virol., 69:1444 [1995]).
  • the nucleotide sequence encoding the Piry G protein is provided in SEQ ID NO:5 and the amino acid sequence of the Piry G protein is provided in SEQ ID NO:6.
  • the nucleotide sequence encoding the Chandipura G protein is provided in SEQ ID NO:7 and the amino acid sequence of the Chandipura G protein is provided in SEQ ID NO:8.
  • the nucleotide sequence encoding the Mokola G protein is provided in SEQ ID NO:9 and the amino acid sequence of the
  • Mokola G protein is provided in SEQ ID NO: 10.
  • Viral particles may be pseudotyped using either the Piry, Chandipura or Mokola G protein as described in Example 2 with the exception that a plasmid containing sequences encoding either the Piry, Chandipura or Mokola G protein under the transcriptional control of a suitable promoter element (e.g., the CMV intermediate- early promoter; numerous expression vectors containing the CMV IE promoter are available, such as the pcDNA3.1 vectors [Invitrogen]) is used in place of pHCMV-G.
  • a suitable promoter element e.g., the CMV intermediate- early promoter; numerous expression vectors containing the CMV IE promoter are available, such as the pcDNA3.1 vectors [Invitrogen]
  • Sequences encoding other G proteins derived from other members of the Rhabdoviridae family may be used; sequences encoding numerous rhabdoviral G proteins are available from the GenBank database.
  • retroviruses which have been shown to infect dividing cells exclusively, or more efficiently, include MLV, spleen necrosis virus, Rous sarcoma virus and human immunodeficiency virus (HIV; while HIV infects dividing cells more efficiently, HIV can infect non-dividing cells).
  • the nuclear envelope of a cell breaks down during meiosis as well as during mitosis. Meiosis occurs only during the final stages of gametogenesis.
  • the methods of the present invention exploit the breakdown of the nuclear envelope during meiosis to permit the integration of recombinant retroviral DNA and permit for the first time the use of unfertilized oocytes (i.e., pre-fertilization and pre-maturation oocytes) as the recipient cell for retroviral gene transfer for the production of transgenic animals. Because infection of unfertilized oocytes permits the integration of the recombinant provirus prior to the division of the one cell embryo, all cells in the embryo will contain the proviral sequences.
  • Oocytes which have not undergone the final stages of gametogenesis are infected with the retroviral vector.
  • the injected oocytes are then permitted to complete maturation with the accompanying meiotic divisions.
  • the breakdown of the nuclear envelope during meiosis permits the integration of the proviral form of the retrovirus vector into the genome of the oocyte.
  • the injected oocytes are then cultured in vitro under conditions which permit maturation of the oocyte prior to fertilization in vitro.
  • Conditions for the maturation of oocytes from a number of mammalian species e.g., bovine, ovine, porcine, murine, caprine
  • the base medium used herein for the in vitro maturation of bovine oocytes may be used for the in vitro maturation of other mammalian oocytes.
  • TC-M199 medium is supplemented with hormones (e.g., luteinizing hormone and estradiol) from the appropriate mammalian species.
  • hormones e.g., luteinizing hormone and estradiol
  • the amount of time a pre-maturation oocyte must be exposed to maturation medium to permit maturation varies between mammalian species as is known to the art. For example, an exposure of about 24 hours is sufficient to permit maturation of bovine oocytes while porcine oocytes require about 44-48 hours.
  • Oocytes may be matured in vivo and employed in place of oocytes matured in vitro in the practice of the present invention.
  • matured pre-fertilization oocytes may be harvested directly from pigs that are induced to superovulate as is known to the art Briefly, on day 15 or 16 of estrus the female pig(s) is injected with about 1000 units of pregnant mare's serum (PMS; available from Sigma and Calbiochem). Approximately 48 hours later, the pig(s) is injected with about 1000 units of human chorionic gonadotropin) (hCG; Sigma) and 24-48 hours later matured oocytes are collected from oviduct.
  • PMS pregnant mare's serum
  • hCG human chorionic gonadotropin
  • in vivo matured pre- fertilization oocytes are then injected with the desired retroviral preparation as described herein.
  • Methods for the superovulation and collection of in vivo matured (i.e., oocytes at the metaphase 2 stage) oocytes are known for a variety of mammals (e.g., for superovulation of mice, see Hogan et al, supra at pp. 130-133 [1994]; for superovulation of pigs and in vitro fertilization of pig oocytes see Cheng, Doctoral Dissertation, Cambridge University,
  • Retroviral vectors capable of infecting the desired species of non-human animal which can be grown and concentrated to very high titers (e.g., > 1 x 10* cfu/ml) are preferentially employed.
  • the use of high titer virus stocks allows the introduction of a defined number of viral particles into the perivitelline space of each injected oocyte.
  • the perivitelline space of most mammalian oocytes can accommodate about 10 picoliters of injected fluid (those in the art know that the volume that can be injected into the perivitelline space of a mammalian oocyte or zygote varies somewhat between species as the volume of an oocyte is smaller than that of a zygote and thus, oocytes can accommodate somewhat less than can zygotes).
  • the vector used may contain one or more genes encoding a protein of interest; alternatively, the vector may contain sequences which produce anti-sense RNA sequences or ribozymes.
  • the infectious virus is microinjected into the perivitelline space of oocytes (including pre-maturation oocytes) or one cell stage zygotes. Microinjection into the perivitelline space is much less invasive than the microinjection of nucleic acid into the pronucleus of an embryo. Pronuclear injection requires the mechanical puncture of the plasma membrane of the embryo and results in lower embryo viability. In addition, a higher level of operator skill is required to perform pronuclear injection as compared to perivitelline injection.
  • the virus stock may be titered and diluted prior to microinjection into the perivitelline space so that the number of proviruses integrated in the resulting transgenic animal is controlled.
  • the use of a viral stock (or dilution thereof) having a titer of 1 x 10' cfu ml allows the delivery of a single viral particle per oocyte.
  • the use of pre-mamration oocytes or mature fertilized oocytes as the recipient of the virus minimizes the production of animals which are mosaic for the provirus as the virus integrates into the genome of the oocyte prior to the occurrence of cell cleavage.
  • the micropipets used for the injection are calibrated as follows.
  • Small volumes (e.g., about 5-10 pi) of the undiluted high titer viral stock (e.g., a titer of about 1 x 10* cfu/ml) are delivered to the wells of a microtiter plate by pulsing the micromanipulator.
  • the titer of virus delivered per a given number of pulses is determined by diluting the viral stock in each well and determining the titer using a suitable cell line (e.g., the 208F cell line) as described in Ex. 2.
  • the number of pulses which deliver, on average, a volume of virus stock containing one infectious viral particle i.e., gives a MOI of 1 when titered on 208F cells) are used for injection of the viral stock into the oocytes.
  • the cumulus cell layer Prior to microinjection of the titered and diluted (if required) virus stock, the cumulus cell layer is opened to provide access to the perivitelline space.
  • the cumulus cell layer need not be completely removed from the oocyte and indeed for certain species of animals (e.g., cows, sheep, pigs, mice) a portion of the cumulus cell layer must remain in contact with the oocyte to permit proper development and fertilization post-injection.
  • Injection of viral particles into the perivitelline space allows the vector RNA (i.e., the viral genome) to enter the cell through the plasma membrane thereby allowing proper reverse transcription of the viral RNA.
  • the presence of the retroviral genome in cells (e.g., oocytes or embryos) infected with pseudotyped retrovirus may be detected using a variety of means.
  • the expression of the gene product(s) encoded by the retrovirus may be detected by detection of mRNA corresponding to the vector-encoded gene products using techniques well known to the art (e.g., Northern blot dot blot in situ hybridization and RT-PCR analysis). Direct detection of the vector-encoded gene product(s) is employed when the gene product is a protein which either has an enzymatic activity (e.g., ⁇ -galactosidase) or when an antibody capable of reacting with the vector- encoded protein is available.
  • an enzymatic activity e.g., ⁇ -galactosidase
  • the presence of the integrated viral genome may be detected using Southern blot or PCR analysis.
  • the presence of the LZRNL or LSRNL genomes may be detected following infection of oocytes or embryos using PCR as follows. Genomic DNA is extracted from the infected oocytes or embryos (the DNA may be extracted from the whole embryo or alternatively various tissues of the embryo may be examined) using techniques well known to the art.
  • the LZRNL and LSRNL viruses contain the neo gene and the following primer pair can be used to amplify a 349-bp segment of the neo gene: upstream primer: 5'-GCATTGCATCAGCCATGATG-3' (SEQ ID NO:l) and downstream primer: 5'- GATGGATTGCACGCAGGTTC-3' (SEQ ID NO:2).
  • upstream primer 5'-GCATTGCATCAGCCATGATG-3' (SEQ ID NO:l)
  • downstream primer 5'- GATGGATTGCACGCAGGTTC-3' (SEQ ID NO:2).
  • the PCR is carried out using well known techniques (e.g., using a GeneAmp kit according to the manufacturer's instructions [Perkin-Elmer]).
  • the DNA present in the reaction is denatured by incubation at 94°C for 3 min followed by 40 cycles of 94°C for 1 min, 60°C for 40 sec and 72°C for 40 sec followed by a final extension at 72°C for 5 min.
  • the PCR products may be analyzed by electrophoresis of 10 to 20% of the total reaction on a 2% agarose gel; the 349-bp product may be visualized by staining of the gel with ethidium bromide and exposure of the stained gel to UN light. If the expected PCR product cannot be detected visually, the D ⁇ A can be transferred to a solid support (e.g., a nylon membrane) and hybridized with a 32 P-labeled neo probe.
  • a solid support e.g., a nylon membrane
  • Southern blot analysis of genomic D ⁇ A extracted from infected oocytes and/or the resulting embryos, offspring and tissues derived therefrom is employed when information concerning the integration of the viral D ⁇ A into the host genome is desired.
  • the extracted genomic D ⁇ A is typically digested with a restriction enzyme which cuts at least once within the vector sequences. If the enzyme chosen cuts twice within the vector sequences, a band of known (ie.,. redictable) size is generated in addition to two fragments of novel length which can be detected using appropriate probes.
  • the present invention also provides transgenic animals that are capable of expressing foreign proteins in their milk, urine and blood.
  • the transgene is stable, as it is shown to be passed from a transgenic bull to his offspring (See, Example 8).
  • transgenic animals produced according to the present invention express foreign proteins in their body fluids (e.g., milk, blood, and urine).
  • body fluids e.g., milk, blood, and urine.
  • Such a promoter could be used to control expression of proteins that would prevent disease and/or infection in the transgenic animals and their offspring, or be of use in the production of a consistent level of protein expression in a number of different tissues and body fluids.
  • the MoMLV LTR of the present invention will find use in driving expression of antibody to pathogenic organisms, thereby preventing infection and/or disease in transgenic animals created using the methods of the present invention.
  • antibodies directed against organisms such as E. coli, Salmonella ssp., Streptococcus ssp., Staphylococcus spp, Mycobacterium spp., produced by transgenic animals will find use preventing mastitis, scours, and other diseases that are common problems in young animals.
  • transgenic animals produced according to the present invention will find use as bacteriostatic, bactericidal, fungistatic, fungicidal, viricidal, and/or anti-parasitic compositions.
  • transgenic animals produced according to the present invention will be resistant to various pathogenic organisms.
  • the milk produced by female transgenic animals would contain substantial antibody levels. It is contemplated that these antibodies will find use in the protection of other animals (e.g., through passive immunization methods).
  • the expression of the fusogenic VSV G protein on the surface of cells results in syncytium formation and cell death. Therefore, in order to produce retroviral particles containing the VSV G protein as the membrane-associated protein a three step approach was taken. First stable cell lines expressing the Gag and Pol proteins from MoMLV at high levels were generated (e.g., 293GP cells; Example 1). These stable cell lines were then infected using the desired retroviral vector which is derived from an amphotrophic packaging cell (e.g., PA317 cells transfected with the desired retroviral vector; Example 2a). The infected stable cell line which expresses the Gag and Pol proteins produces noninfectious viral particles lacking a membrane-associated protein (e.g., a envelope protein).
  • a membrane-associated protein e.g., a envelope protein
  • these infected cell lines are then transiently transfected with a plasmid capable of directing the high level expression of the VSV G protein (Example 2b).
  • the transiently transfected cells produce VSV G-pseudotyped retroviral vectors which can be collected from the cells over a period of 3 to 4 days before the producing cells die as a result of syncytium formation.
  • the first step in the production of VSV G-pseudotyped retroviral vectors, the generation of stable cell lines expressing the MoMLV Gag and Pol proteins is described below.
  • the human adenovirus 5-transformed embryonal kidney cell line 293 (ATCC CRL 1573) was cotransfected with the pCMVgag-pol and pFR400 plasmids using a ratio of 10:1
  • pCMVgag-pol and pFR400 contains the MoMLV gag and pol genes under the control of the CMV promoter (pCMV gag-pol is available from the ATCC).
  • pFR400 encodes a mutant dihydrofolate reductase which has a reduced affinity for methotrexate (Simonsen et al, Proc. Natl. Acad. Sci. 80:2495 [1983]).
  • the plasmid DNA was introduced into the 293 cells using calcium phosphate co- precipitation (Graham and Van der Eb, Virol., 52:456 [1973]).
  • VSV G protein pseudotyped retrovirus In order to produce VSV G protein pseudotyped retrovirus the following steps were taken. First die 293 GP cell line was infected with virus derived from the amphotrophic packaging cell line PA317. The infected cells packaged the retroviral RNA into viral particles which lack a membrane-associated protein (because the 293GP cell line lacks an env gene or other gene encoding a membrane-associated protein). The infected 293 GP cells were then transiently transfected with a plasmid encoding the VSV G protein to produce pseudotyped viral particles bearing the VSV G protein.
  • the amphotropic packaging cell line, PA317 (ATCC CRL 9078) was grown in DMEM-high glucose medium containing 10% FCS.
  • the 293GP cell line was grown in DMEM-high glucose medium containing 10% FCS.
  • the titer of the pseudo-typed virus may be determined using either 208F cells (Quade, Virol., 98:461 [1979]), or NIH/3T3 cells (ATCC CRL 1658); 208F and NIH 3T3 cells are grown in DMEM-high glucose medium containing 10% CS.
  • the plasmid pLZRNL (Xu et al., Virol., 171:331 [1989]) contains the gene encoding E. coli ⁇ -galactosidasc (LacZ) under the transcriptional control of the LTR of the Moloney murine sarcoma virus (MSV) followed by the gene encoding neomycin phosphotransferase (Neo) under the transcriptional control of the Rous sarcoma virus (RSV) promoter.
  • E. coli ⁇ -galactosidasc (LacZ) under the transcriptional control of the LTR of the Moloney murine sarcoma virus (MSV) followed by the gene encoding neomycin phosphotransferase (Neo) under the transcriptional control of the Rous sarcoma virus (RSV) promoter.
  • the plasmid pLSRNL contains the gene encoding the hepatitis B surface antigen gene (HBsAg) under the transcriptional control of the MSV LTR followed by the Neo gene under the control of the RSV promoter (U.S. Patent No. 5,512,421, the disclosure of which is herein incorporated by reference).
  • the plasmid pHCMV-G contains the VSV G gene under the transcriptional control of the human cytomegalovirus intermediate-early promoter (Yee et al. Meth. Cell Biol., 43:99 [1994]).
  • PA317 cells were placed in a 100 mm tissue culture plate.
  • day 2 the PA317 cells were transfected with 20 ⁇ g of pLZRNL plasmid DNA (plasmid DNA was purified using CsCl gradients) using the standard calcium phosphate co-precipitation procedure (Graham and Van der Eb, Virol., 52:456 [1973]).
  • plasmid DNA was purified using CsCl gradients
  • a range of 10 to 40 ⁇ g of plasmid DNA may be used.
  • 293GP cells may take more than 24 hours to attach firmly to tissue culture plates, the 293GP cells may be placed in 100 mm plates 48 hours prior to transfection.
  • the transfected PA317 cells provide amphotropic LZRNL virus.
  • the culture medium containing LZRNL virus (containing polybrene) was used to infect the 293GP cells as follows. The culture medium was removed from the 293 GP cells and was replaced with the LZNRL virus containing culture medium. The virus containing medium was allowed to remain on the
  • 293 GP cells for 16 hours. Following the 16 hour infection period (on day 5), the medium was removed from the 293GP cells and was replaced with fresh medium containing 400 ⁇ g/ml G418 (GIBCO/BRL). The medium was changed every 3 days until G418-resistant colonies appeared two weeks later. Care was taken not to disturb the G418-resistant colonies when the medium was changed as 293GP cells attach rather loosely to tissue culture plates. The G418-resistant 293 colonies were picked using an automatic pipettor and transfe ⁇ ed directly into 24-well plates (i.e., the colonies were not removed from the plates using trypsin).
  • G418-resistant 293 colonies were picked using an automatic pipettor and transfe ⁇ ed directly into 24-well plates (i.e., the colonies were not removed from the plates using trypsin).
  • the G418-resistant 293 colonies (as termed "293GP/LZRNL” cells) were screened for the expression of the LacZ gene in order to identity clones which produce high titers of pseudotyped LZRNL virus.
  • Clones in 24-well plates were transferred to 100 mm tissue culture plates and allowed to grow to confluency. Protein extracts are prepared from the confluent plates by washing the cells once with 10 ml PBS (137 mM NaCl, 2.6 mM KCl, 8.1 mM Na_HPO 4 , 1.5 mM KH 2 PO 4 ). Two ml of 250 mM Tris-HCl, pH 7.8 was added and the cells were scrapped off the plate using a rubber policeman.
  • the cells were then collected by centrifugation at room temperature and resuspended in 100 ⁇ l 250 mM Tris-HCl, pH 7.8. The cells were subjected to four rapid freeze/thaw cycles followed by centrifugation at room temperature to remove cell debris.
  • the ⁇ -galactosidase activity present in the resulting protein extracts was determined as follows. Five microliters of protein extract was mixed with 500 ⁇ l ⁇ -gal buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl j ) containing 0.75 ONPG (Sigma). The mixtures were incubated at 37°C until a yellow color appeared. The reactions were stopped by the addition of 500 ⁇ l 10 mM EDTA and the optical density of the reactions was determined at 420 nm.
  • the 293GP LZRNL clone which generated the highest amount of ⁇ -galactosidase activity was then expanded and used subsequently for the production of pseudotyped LZNRL virus as follows. Approximately 1 x 10 6 293GP/LZRNL cells were placed into a 100 mm tissue culture plate. Twenty-four hours later, the cells were transfected with 20 ⁇ g of pHCMV-G plasmid DNA using calcium phosphate co-precipitation. Six to eight hours after the calcium-DNA precipitate was applied to the cells, the DNA solution was replaced with fresh culture medium (lacking G418).
  • the pseudotyped LZRNL virus generated from the transfected 293GP/LZRNL cells can be collected at least once a day between 24 and 96 hr after transfection.
  • the highest virus titer was generated approximately 48 to 72 hr after initial pHCMV-G transfection. While syncytium formation became visible about 48 hr after transfection in the majority of the transfected cells, the cells continued to generate pseudotyped virus for at least an additional 48 hr as long as the cells remained attached to the tissue culture plate.
  • the collected culture medium containing the VSV G-pseudotyped LZRNL virus was pooled, filtered through a 0.45 ⁇ m filter and stored at -70°C.
  • the titer of the VSV G-pseudotyped LZRNL virus was then determined as follows. 5 x 10 s rat 208F fibroblasts or NIH 3T3 cells were plated in a 100 mm culture plate. Twenty- fours hours after plating* the cells were infected with serial dilutions of the LZRNL virus- containing culture medium in the presence of 8 ⁇ g/ml polybrene. Sixteen hours after infection with virus, the medium was replaced with fresh medium containing 400 ⁇ g/ml G418 and selection was continued for 14 days until G418-resistant colonies became visible. Viral titers were typically about 0.5 to 5.0 x 10* colony forming units (cfu)/ml. The titer of the virus stock could be concentrated to a titer of greater than 10 9 cfu/ml as described below.
  • the VSV G-pseudotyped LZRNL virus was concentrated to a high titer by two cycles of ultracentrifugation.
  • the frozen culture medium collected as described in Example 2 which contained pseudotyped LZRNL virus was thawed in a 37°C water bath and was then transferred to ultraclear centrifuge tubes (14 x 89 mm; Beckman, Palo Alto, CA) which had been previously sterilized by exposing the tubes to UV light in a laminar flow hood overnight.
  • the virus was sedimented in a SW41 rotor (Beckman) at 50,000 x g (25,000 rpm) at 4°C for 90 min.
  • the culture medium was then removed from the tubes in a laminar flow hood and the tubes were well drained.
  • the virus pellet was resuspended to 0.5 to 1% of the original volume of culture medium in either TNE (50 mM Tris-HCl, pH 7.8; 130 mM NaCl; 1 mM EDTA) or 0.1 X Hank's balanced salt solution (IX Hank's balanced salt solution contains 1.3 mM CaCl 2 , 5 mM KCl, 0.3 mM KH 2 PO 4 , 0.5 mM MgCl 2 « 6H 2 ), 0.4 mM MgSO 4 » 7H 2 O, 138 mM NaCl, 4 mM NaHCO 3 , 0.3 mM NaH 2 PO 4 « H 2 O; 0.1X Hank's is made by mixing 1 parts IX Hank's with 9 parts PBS].
  • the resuspended virus pellet was incubated overnight at 4°C without swirling.
  • the virus pellet could be dispersed with gentle pipetting after the overnight incubation without significant loss of infectious virus.
  • the titer of the virus stock was routinely increase 100- to 300-fold after one round of ultracentrifugation. The efficiency of recovery of infectious virus varied between 30 and 100%.
  • the virus stock was then subjected to low speed centrifugation in a microfuge for 5 min at 4°C to remove any visible cell debris or aggregated virions that were not resuspended under the above conditions (if the virus stock is not to be used for injection into oocytes or embryos, this centrifugation step may be omitted).
  • the virus stock was then subjected to another round of ultracentrifugation to concentrate the virus stock further.
  • the resuspended virus from the first round of centrifugation was pooled and pelleted by a second round of ultracentrifugation which was performed as described above.
  • Viral titers were increased approximately 2000-fold after the second round of ultracentrifugation (titers of the pseudotyped LZRNL virus were typically greater than or equal to 1 x 10' cfu/ml after the second round of ultracentrifugation).
  • the titers of the pre- and post-centrifugation fluids were determined by infection of
  • 208F NASH 3T3 or Mac-T cells can also be employed
  • G418-resistant colonies as described above in Example 2.
  • the concentrated viral stock was stable (i.e., did not lose infectivity) when stored at 4°C for several weeks.
  • the concentrated pseudotyped retrovirus were resuspended in 0.1 X HBS (2.5 mM HEPES, pH 7.12, 14 mM NaCl, 75 ⁇ M Na 2 HPO 4 « H 2 O) and 18 ⁇ l aliquots were placed in 0.5 ml vials (Eppendorf) and stored at -80°C until used.
  • the titer of the concentrated vector was determined by diluting l ⁇ l of the concentrated virus 10 "7 - or 10 "8 -fold with 0.1X HBS.
  • the diluted virus solution was then used to infect 208F and Mac-T cells and viral titers were determined as described in Example 2.
  • polybrene Prior to infection of oocytes or embryos (by microinjection), 1 ⁇ l of polybrene (25 ng/ ⁇ l; the working solution of polybrene was generated by diluting a stock solution having a concentration of 1 mg/ml [in sterile H 2 O], in 0.1 HBS, pH 7.12) was mixed with 4 ⁇ l of concentrated virus to yield a solution containing 10M0 4 cfu/ ⁇ l and 8 ⁇ g/ml polybrene. This solution was loaded into the injection needle (tip having an internal diameter of approximately 2-4 ⁇ m) for injection into the perivitelline space of gametes (pre-maturation oocytes, matured oocytes) or one cell stage zygotes (early stage embryo). An Eppendorf Transjector 5246 was used for all microinjections.
  • Gametes pre-maturation and pre-fertilization oocytes
  • zygotes fertilized oocytes
  • Tyrodes-Lactate with HEPES 114 mM NaCl, 3.2 mM KCl, 2.0 mM NaHCO 3 , 0.4 mM 10 mM Na-lactate, 2 mM CaCl 2 '2H_O, 0.5 mM MgCl 2 » 6H 2 O, 10 mM HEPES, 100 IU/ml penicillin, 50 ⁇ g/ml phenol red, 1 mg/ml BSA fraction V, 0.2 mM pyruvate and 25 ⁇ g/ml gentamycin.
  • Maturation Medium TC-199 medium (GIBCO) containing 10% FCS, 0.2 mM pyruvate, 5 ⁇ g/ml NIH o-LH (NIH), 25 ⁇ g/ml gentamycin and 1 ⁇ g/ml estradiol-17 ⁇ .
  • Sperm-Tyrodes-Lactate 100 mM NaCl, 3.2 mM KCl, 25 mM NaHCO 3 , 0.29 mM Na 2 H 2 PO 4 « H 2 O, 21.6 mM Na-lactate, 2.1 mM CaCl 2 » 2H 2 O, 0.4 mM MgCl 2 »6H 2 O,
  • Fertilization Medium 114 mM NaCl, 3.2 mM KCl, 25 mM NaHCO 3 , 0.4 mM Na 2 H 2 PO 4 ⁇ 2 O, 10 mM Na-lactate, 2 mM CaCl 2 « 2H 2 O, 0.5 mM MgCl 2 » 6H 2 O, 100 IU/ml penicillin, 50 ⁇ g/ml phenol red, 6 mg/ml BSA fatty acid free, 0.2 mM pyruvate and 25 ⁇ g/ml gentamycin.
  • PHE 1 mM hypotaurine, 2 mM penicillamine and 250 ⁇ M epinephrine.
  • EIAA Embryo Incubation + Amino Acids
  • 0.1X HBS 2.5 mM HEPES (pH 7.12), 14 mM NaCl and 75 ⁇ M
  • Oocytes were aspirated from small antral follicles on ovaries from dairy cattle obtained from a slaughterhouse. Freshly aspirated oocytes at the germinal vesicle (GV) stage, meiosis arrested, with the cumulus mass attached were selected (i.e., pre-maturation oocytes). The oocytes were then washed twice in freshly prepared TL-HEPES and transferred into a 100 ⁇ l drop of TL-HEPES for microinjection.
  • GV germinal vesicle
  • Concentrated retroviral particles (prepared as described in Example 3) were resuspended in 0.1X HBS, mixed with polybrene and loaded into the injection needle as described in Example 4. Approximately 10 pi of the virus solution was then injected into the perivitelline space of pre-maturation oocytes.
  • the pre-maturation oocytes were washed twice in fresh TL-HEPES and transferred into maturation medium (10 oocytes in 50 ⁇ l). The pre-maturation oocytes were then incubated in Maturation Medium for 24 hours at 37°C which permits the oocytes to mature to the metaphase II stage. The matured oocytes were then washed twice insperm-TL and 10 oocytes were then transferred into 44 ⁇ l of Fertilization Medium. The mature oocytes
  • Frozen-thawed semen was layered on top of the gradient and the tubes were centrifuged for 10 minutes at 700xg. Motile sperm were collected from the bottom of the tube.
  • the oocytes were incubated for 16 to 24 hours at 37°C in the fertilization mixture. Following fertilization, the cumulus cells were removed by vortexing the cells (one cell stage zygotes, Pronucleus Stage) for 3 minutes to produce "nude" oocytes.
  • the nude oocytes were then washed twice in embryo culture medium (EIAA) and 20 to 25 zygotes were then cultured in 50 ⁇ l drop of EIAA (without serum until Day 4 at which time the zygotes were placed in EIAA containing 10% serum) until the desired developmental stage was reached: approximately 48 hours or Day 2 (Day 0 is the day when the matured oocytes are co-cultured with sperm) for morula stage (8 cell stage) or Day 6-7 for blastocyst stage. Embryos at the morula stage were analyzed for expression of ⁇ -galactosidase as described in Example 6. Embryos derived from injected pre-maturation oocytes were also analyzed for ⁇ -galactosidase expression at the 2 cell, 4 cell, and blastocyst stage and all developmental stages examined were positive.
  • EIAA embryo culture medium
  • Pre-Fertilization Oocytes Pre-maturation oocytes were harvested, washed twice with TL-HEPES as described above. The oocytes were then cultured in Maturation Medium (10 oocytes per 50 ⁇ l medium) for 16 to 20 hours to produce pre-fertilization oocytes (Metaphase II Stage). The pre-fertilization or matured oocytes were then vortexed for 3 minutes to remove the cumulus cells to produce nude oocytes. The nude oocytes were washed twice in TL-HEPES and then transferred into a 100 ⁇ l drop of TL-HEPES for microinjection. Microinjection was conducted as described above. Following microinjection, the pre-fertilization oocytes were washed twice with TL-
  • Microinjection of retrovirus was conducted as described above. Following microinjection, the zygotes were washed four times in EIAA and then placed in an EIAA culture drop (25 zygotes per 50 ⁇ l drop of EIAA). The zygotes were cultured in EIAA (20 to 25 zygote per 50 ⁇ l drop of EIAA) until the desired developmental stage was reached. The embryos were then examined for ⁇ -galactosidase expression (Ex. 6) or transferred to recipient cows (Ex. 7).
  • Oocytes and one-cell zygotes which had been microinjected with pseudotyped LZRNL virus and cultured in vitro were examined for expression of vector sequences by staining for ⁇ -galactosidase activity when the embryos had reached the morula stage, ⁇ -galactosidase activity was examined as follows. Embryos were washed twice in PBS then fixed in 0.5% glutaraldehyde in PBS containing 2mM MgCl 2 for 40 min. at 4°C .
  • the methods of the present invention provide an efficiency of generating transgenic embryos which is superior to existing methods.
  • Embryos derived from infected pre-fertilization oocytes and early zygotes were transferred into recipient cows which were allowed to progress to term as described below.
  • Embryos Derived From Infected Oocytes and Zygotes Pre-fertilization oocytes (infected about 17 hours after exposure to Maturation Medium) and early stage zygotes ( ⁇ 8 cell stage) were prepared and infected as described in Example 5 with the exceptions that 1) the VSV-G-pseudotyped virus used was the LSRNL virus which was prepared as described for the LZRNL virus in Ex. 2, and 2) at day 4 post- fertilization, embryos derived from injected pre-fertilization oocytes and zygotes were placed in freshly prepared EIAA medium containing 10% FCS and allowed to develop in vitro until transfer into recipient cows. Embryos at Day 7 were transferred into recipient females which were prepared as described below.
  • Recipient cows were synchronized by injecting 100 ⁇ g of gonadotropin-releasing hormone (GnRH; Sanofi Winthrop Pharmaceutical Inc., New York, NY) (Day 0). Seven days later, the recipients were injected with 25 mg of PGF2 ⁇ (Upjohn Co., Kalamazoo, MI).
  • GnRH gonadotropin-releasing hormone
  • PGF2 ⁇ Upjohn Co., Kalamazoo, MI
  • 8 embryos derived from infected pre-fertilization oocytes were transferred into 4 recipients; four calves were born to these recipients and all four were found to be positive for the presence of vector proviral DNA (i.e., 100% were transgenic).
  • 8 embryos derived from post-fertilization zygotes were transferred into 4 recipients; 2 calves were born to these recipients and one of these animals was found to be transgenic (in the second transfer, one pregnancy was lost in the first month and another pregnancy comprising twins was lost in the eighth month; neither embryo from the 8 month pregnancy was transgenic).
  • the third transfer 4 embryos derived from infected zygotes infected at the 4-8 cell stage were transferred into 2 recipients; 3 calves were born to these recipients and none were transgenic.
  • GEBCO proteinase K
  • the DNA present in the aqueous layer was then precipitated by the addition of 2 volumes of isopropanol.
  • the DNA was collected by centrifugation and the DNA pellet was resuspended in TE buffer (lOmM Tris-Cl, 1 mM EDTA, pH 8.0) and the concentration was determined spectrophotometrically.
  • the DNA was then analyzed by Southern blotting and PCR analysis.
  • Figure 2 shows an autoradiography of a Southern blot of genomic DNA isolated from the skin (Fig. 2A) and blood (Fig. 2B) of the six calves derived from either pre-fertilization oocytes infected with VSV G-pseudotyped LSRNL virus at about 17 hours after exposure to Maturation Medium (calves numbered 17, 18, 20 and 21) or one cell zygotes infected at about
  • Lanes labelled "LSRNL pDNA” contain Hi ⁇ dlll -digested pLSRNL plasmid DNA and provide controls for the quantitation of the copy number of the integrated proviruses in the offspring (DNA equivalent to 5, 10 or 25 copies of LSRNL were applied in these lanes).
  • ⁇ BsAG probe was generated by PCR amplification of pLSRNL plasmid DNA using the upstream primer S-l (5'-GGCTATCGCTGGATGTGTCT-3'; [SEQ ID NO:3]) and the downstream primer S-3 (5'-ACTGAACAAATGGCACTAGT-3'; [SEQ ID NO:4]).
  • the PCR- generated probe (334 bp) was labeled using a Rediprime kit (Amersham, Arlington Heights, IL) according to the manufacturer's instructions.
  • the autoradiographs shown in Fig. 2 were generated by exposure of the blots to X-ray film for 3 weeks at -80°C.
  • the results shown in Figure 2 demonstrates that calves 16, 17, 18, 20 and 21 contained retroviral vector DNA in both the skin (Fig. 2A) and blood (Fig. 2B).
  • blood cells (buffy coat) are derived from the mesoderm and skin cells are derived from the ectoderm, these results show that the transgenic animals do not display somatic cell mosaicism.
  • Figure 3 shows the results of the PCR analysis following amplification of two different regions (i.e., the neo gene and the HBsAg gene) of the LZRNL retroviral genome which was injected into the oocytes.
  • Genomic DNA from the skin and blood of each of the five transgenic calves was amplified using the upstream and downstream primers (SEQ ID NOS:l and 2 and NOS:3 and 4; described supra) for the neo (Fig. 3A) and HBsAg (Fig. 3B) genes, respectively.
  • the PCRs were conducted using the following thermocycling conditions: 94°C ( 4 min); (94°C [2 min]; 50°C [2 min];
  • transgenic animals which contain a single copy of the transgene.
  • the transgenic offspring are bred with non-transgenic cattle and the presence of the viral sequences (i.e., the transgene) determined using Southern blot analysis or PCR amplification as described above. Animals which are heterozygous or homozygous for the transgene are produced using methods well known to the art (e.g., interbreeding of animals heterozygous for the transgene).
  • transgenic bulls produced either by perivitelline space injection of an unfertilized oocyte (#21) or by perivitelline space injection of a fertilized zygote (#16) have the transgene present in their sperm, and are thus capable of passing the transgene on to their offspring. Indeed, as described in Example 9 below, bull #16 has produced two live transgenic offspring.
  • transgenic bull designated as #16
  • a transgenic bull produced through one-cell zygotic injection, was naturally mated with a non-transgenic cow. This mating resulted in the production of twin calves, one female (designated " as #42) and one male (designated as #43). Blood and skin samples were taken from each of the calves, and their DNA was isolated using methods known in the art (See e.g., Hogan et al frequent Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY [1986]).
  • PCR was performed on these DNA samples, using the methods described in Example 7, above.
  • Two sets of primers were used to analyze both the blood and skin samples.
  • One set of primers (“Neol” and “Neo2”) was used to detect the neomycin resistance gene in the LSRNL vector. The location and description of these primers is shown in Figure 3 A.
  • the second set of primers (SI and S3) were used to detect a portion of the Hepatitis B surface antigen (HBsAg) gene in the LSRNL vector. The location and description of these primers is shown in Figure 3B.
  • FIG. 4 shows the results of PCR screening of skin samples from these calves.
  • the control animal is indicated as #12, while the offspring are indicated as #42 and #43, as described above.
  • Lane one contains DNA size standards, and lanes 2-4 contain the DNA samples analyzed using the Meo PCR primers, while lanes 5-7 contain the same DNA samples analyzed using the HBsAg PCR primers.
  • the correct size for the neo band is 349 base pairs, while the correct size for the HBsAg band is slightly smaller, at 334 base pairs.
  • transgenic animals can be successfully created by perivitelline space injection of a one-cell zygote with a pseudotyped replication-defective retrovirus.
  • these data demonstrate that the incorporated transgene is passed on the offspring of the transgenic animal.
  • Milk samples were collected from #17 and #18, and five control heifers that had also been artificially induced to lactate using the same protocol and at the same time as #17 and #18. Whole milk samples were analyzed using the AUSZYME® Monoclonal Antibody Assay (Abbott), for the detection of HBsAg. The milk samples collected from #17 and #18 tested positive for HBsAg. The milk samples from the five control heifers were all negative for the antigen.
  • Abbott AUSZYME® Monoclonal Antibody Assay
  • the estimated level of HBsAg production was found to be 200 ng HBsAg/ml milk, for #17, and 700 ng HBsAg/ml milk, for #18.
  • HBsAg blood and urine samples were also collected from the two female founder transgenic heifers #17 and #18.
  • the serum was separated from the whole blood using methods known in the art (i.e., centrifugation).
  • the urine and serum samples were assayed for the presence of HBsAg using the AUSZYME® system, as per the kit manufacturer's instructions.
  • the urine and serum of #17 and #18 all tested positive for the presence of HBsAg, while the urine and serum samples from the control animals all tested negative. Based on this test system, the estimated level of HBsAg production for #17 was 2.58 ng HBsAg/ml of serum, and 0.64 ng HBsAg/ml of urine.
  • the values were 0.64 ng HBsAg/ml of serum, and 0.97 ng HBsAg/ml of urine. These data demonstrate that transgenic animals produced by perivitelline space injection of an unfertilized oocyte are capable of producing substantial levels of foreign proteins in their serum and urine. These data further demonstrate the utility of using the MoMLV LTR as a promoter for driving the constitutive production of foreign proteins in transgenic cattle, as this promoter was shown in these experiments to cause the production of
  • HBsAg in milk, serum, and urine of transgenic cattle.
  • the term “constitutive” refers to a relatively low level of expression throughout the animal's body.
  • the term “preferentially expressed” indicates that a relatively high level of expression is achieved in certain tissues or body fluids, as compared to other tissues and fluids. For example, in preferred embodiments of the present invention, foreign proteins of interest are preferentially expressed in such fluids as milk.
  • Such a promoter could be used to control expression of proteins that would prevent disease and/or infection in the transgenic animals and their offspring, or be of use in the production of a consistent level of protein expression in a number of different tissues and body fluids.
  • the invention provides improved methods and compositions for the production of transgenic non-human animals.
  • the methods of the present invention provide for the production of transgenic non-human animals with improved efficiency and a reduced incidence of generating animals which are mosaic for the presence of the transgene.
  • NAME Ingolia, Diane E.
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • DESCRIPTION: /desc "DNA”
  • FEATURE FEATURE
  • GCT CTC CAA AGG TAT AAA GAT GGG AGC TTA ATC AAT CTT GGA TTC CCC 384 Ala Leu Gin Arg Tyr Lys Asp Gly Ser Leu lie Asn Leu Gly Phe Pro 115 120 125
  • Trp lie Asp Pro Leu Phe Pro Gly Gly Glu Cys Ser Thr Asn Phe Cys 165 170 175
  • Trp He Asp Pro Leu Phe Pro Gly Gly Glu Cys Ser Thr Asn Phe Cys 165 170 175 Asp Thr Val His Asn Ser Ser Val Trp He Pro Lys Ser Gin Lys Thr 180 185 190
  • MOLECULE TYPE other nucleic acid
  • GCT CTC CAA AGG TAT AAA GAT GGG AGC TTA ATC AAT CTT GGA TTC CCC 384 Ala Leu Gin Arg Tyr Lys Asp Gly Ser Leu He Asn Leu Gly Phe Pro 115 120 125
  • MOLECULE TYPE other nucleic acid
  • GTC ACC ACC ACC TTC AAA AGG AAG CAC TTT AAA CCT ACA GTG GCT GCT 336 Val Thr Thr Thr Phe Lys Arg Lys His Phe Lys Pro Thr Val Ala Ala 100 105 110
  • Trp Glu Ser Ser Lys Gly Leu Pro Gly Thr 515 520

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CHAN A W S ET AL: "Transgenic cattle produced by reverse-transcribed gene transfer in oocyte", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 95, no. 24, 24 November 1998 (1998-11-24), pages 14028 - 14033, XP002173620, ISSN: 0027-8424 *
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