Multicellular living organisms and unmodified parts thereof and – Method of making a transgenic nonhuman animal – Via microinjection of a nucleus into an embryo – egg cell – or...
Reexamination Certificate
1999-04-23
2003-06-17
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of making a transgenic nonhuman animal
Via microinjection of a nucleus into an embryo, egg cell, or...
C800S008000, C800S014000
Reexamination Certificate
active
06580017
ABSTRACT:
BACKGROUND OF THE INVENTION
Recently, developments in the area of cloning and cloning procedures have expanded. For example, there have been several reports of live births of animals using cloning procedures. Live lambs were produced following nuclear transfer of cultured embryonic disc cells. Campbell et al. (1996)
Nature
380:64-68. Campbell et al. disclose methods of transferring the nucleus of a quiescent cell into an enucleated oocyte to form a reconstructed embryo. The reconstructed embryo was developed until the morula to blastocyst stage in vivo prior to transfer into a recipient female.
Other reports of the live births of cloned mammals have also relied upon the transfer of a reconstructed embryo after it has reached the blastocyst stage of embryogenesis.
SUMMARY OF THE INVENTION
The present invention is based, in part, on the discovery that a reconstructed embryo which is transferred into a recipient mammal at the two to four cell stage of embryogenesis can develop into a cloned mammal. The mammal can be an embryo, a fetus, or a post natal mammal, e.g., an adult mammal.
Accordingly, in one aspect, the invention features a method of producing a non-human mammal, e.g., a cloned mammal, e.g., a goat, cow, pig, horse, sheep, llama, camel. The method includes maintaining a mammalian reconstructed embryo, e.g., a reconstructed embryo wherein the genome is derived from a somatic cell, in culture until the embryo is in the 2 to 8 cell stage, transferring the embryo at the 2 to 8 cell stage into a recipient mammal, and allowing the reconstructed embryo to develop into a mammal, to thereby produce a mammal.
In a preferred embodiment, the mammal develops from the reconstructed embryo. In another embodiment, the mammal is a descendant of a mammal which developed from the reconstructed embryo.
In a preferred embodiment, the reconstructed embryo is maintained in culture until the embryo is in the 2 to 8, the 2 to 6, the 2 to 4 cell stage of embryogenesis.
In a preferred embodiment, the genome of the reconstructed embryo is derived from: a somatic cell, e.g., a fibroblast or epithelial cell; a genetically engineered somatic cell, e.g., a somatic cell comprising a transgenic sequence.
In a preferred embodiment, the method further includes mating the mammal which develops from the reconstructed embryo with: a second mammal; a second mammal which develops from a reconstructed embryo or is descended from a mammal which developed from a reconstructed embryo; or a second mammal developed from a reconstructed embryo, or descended from a mammal which developed from a reconstructed embryo, which was formed from genetic material from the same animal, an animal of the same genotype, or same cell line, which supplied the genetic material for the first mammal. In a preferred embodiment, a first transgenic mammal which develops from the reconstructed embryo can be mated with a second transgenic mammal which developed from a reconstructed embryo and which contains a different transgene that the first transgenic mammal.
In a preferred embodiment, the mammal is a male mammal. In other preferred embodiments, the mammal is a female mammal. A female mammal can be induced to lactate and milk can be obtained from the mammal.
In a preferred embodiment: a product, e.g., a protein, e.g., a recombinant protein, e.g., a human protein, is recovered from the mammal; a product, e.g., a protein, e.g., a human protein, is recovered from the milk, urine, hair, blood, skin or meat of the mammal.
In a preferred embodiment, the mammal is: embryonic; fetal; or, postnatal, e.g., adult.
In a preferred embodiment, the genome of the reconstructed embryo is derived from a genetically engineered somatic cell, e.g., a transgenic cell or a cell which a nucleic acid has been introduced.
In another aspect, the invention features a method of producing a non-human mammal, e.g., a transgenic mammal, e.g., a goat, cow, pig, horse, sheep, llama, camel. The method includes maintaining a mammalian reconstructed embryo (e.g., a reconstructed embryo wherein its genome is derived from a genetically engineered somatic cell) in culture until the embryo is in the 2 to 8 cell stage, transferring the embryo at the 2 to 8 cell stage into a recipient mammal, and allowing the reconstructed embryo to develop into a mammal, to thereby produce a transgenic mammal.
In a preferred embodiment, the mammal develops from the reconstructed embryo. In another embodiment, the mammal is a descendant of a mammal which developed from the reconstructed embryo.
In a preferred embodiment, the reconstructed embryo is maintained in culture until the embryo is in the 2 to 8, the 2 to 6, the 2 to 4 cell stage of embryogenesis.
In a preferred embodiment, the method further includes mating the mammal which develops from the reconstructed embryo with: a second mammal; a second mammal which develops from a reconstructed embryo or is descended from a mammal which developed from a reconstructed embryo; or a second mammal developed from a reconstructed embryo, or descended from a mammal which developed from a reconstructed embryo, which was formed from genetic material from the same animal, an animal of the same genotype, or same cell line, which supplied the genetic material for the first mammal. In a preferred embodiment, a first transgenic mammal which develops from the reconstructed embryo can be mated with a second transgenic mammal which developed from a reconstructed embryo and which contains a different transgene that the first transgenic mammal.
In a preferred embodiment, the mammal is a male mammal. In other preferred embodiments, the mammal is a female mammal. A female mammal can be induced to lactate and milk can be obtained from the mammal.
In a preferred embodiment: a product, e.g., a protein, e.g., a recombinant protein, e.g., a human protein, is recovered from the mammal; a product, e.g., a protein, e.g., a human protein, is recovered from the milk, urine, hair, blood, skin or meat of the mammal.
In a preferred embodiment, the genome of the genetically engineered somatic cell includes a transgenic sequence. The transgenic sequence can be any of: a heterologous transgene, e.g., a human transgene; a knockout, knockin or other event which disrupts the expression of a mammalian gene; a sequence which encodes a protein, e.g., a human protein; a heterologous promoter; a heterologous sequence under the control of a promoter, e.g., a caprine promoter. The transgenic sequence can encode any product of interest such as a protein, polypeptide or peptide.
In a preferred embodiment, the transgenic sequence encodes any of: a hormone, an immunoglobulin, a plasma protein, and an enzyme. The transgenic sequence can encode any protein whose expression in the transgenic mammal is desired, e.g., any of: &agr;-1 proteinase inhibitor, alkaline phosphotase, angiogenin, extracellular superoxide dismutase, fibrogen, glucocerebrosidase, glutamate decarboxylase, human serum albumin, myelin basic protein, proinsulin, soluble CD4, lactoferrin, lactoglobulin, lysozyme, lactoalbumin, erythrpoietin, tissue plasminogen activator, human growth factor, antithrombin III, insulin, prolactin, and (&agr;1-antitrypsin.
In a preferred embodiment, the transgenic sequence encodes a human protein.
In a preferred embodiment, the transgenic sequence is under the control of a promoter, e.g., a caprine or heterologous promoter. The promoter can be a tissue-specific promoter. The tissue specific promoter can be any of: milk-specific promoters; blood-specific promoters; muscle-specific promoters; neural-specific promoters; skin-specific promoters; hair-specific promoters; and urine-specific promoters. The milk-specific promoter can be, e.g., any of: a casein promoter, a beta lactoglobulin promoter, a whey acid protein promoter and a lactalbumin promoter.
In a preferred embodiment, a nucleic acid can be introduced into the genome of the genetically engineered somatic cell. The nucleic acid can be any of: a heterologous transgene, e.g., a human transgene; a knockout, knockin or other event which disrupts th
Behbodi Esmail
Echelard Yann
Gavin William
Melican David
Crouch Deborah
Fish & Richardson P.C.
Genzyme Transgenics Corporation
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