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
2001-01-10
2003-10-21
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...
C435S375000, C435S377000, C435S384000
Reexamination Certificate
active
06635802
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of mammalian cell biology. More particularly, it concerns methods and compositions using apoptosis inhibitors to increase the efficiency of nuclear transfer, useful in the production of transgenic and non-transgenic mammals from cultured cells or cell lines. Methods using apoptosis inhibitors in cloning mammals, and for producing chimeric cell lines, transgenic and chimeric mammalian tissues and mammals are also provided.
2. Description of Related Art
The basic procedure for nuclear transfer concerns obtaining single cells and fusing them to enucleated recipient ovum. This effectively transfers the nucleus of the donor cell into the recipient cytoplasm where, if successful, it is reprogrammed and subsequently instructs development of a new embryo that is genetically identical to that from which the cell was acquired. Nuclei from embryonic fibroblasts as well as adult mammary epithelial cells can direct normal development in the sheep (Wilmut et al., 1997).
Although the nuclear transfer technique is less advanced in pigs, there have been reports of successful births using nuclei from 4-cell embryos (Prather et al., 1989). Primordial germ cells (PGCs) collected from fetal tissue have also been successfully utilized as donors for nuclear transplantation (Cherny and Merei, 1994, Delhaise et al., 1995, Lavoir et al., 1997, Strelchenko, 1996). In pigs it has been demonstrated that previously cryopreserved PGCs can be used successfully as nuclear donors, giving rise to nuclear reprogramming and cleavage to the 4-cell stage (Liu et al., 1995). Additionally, nuclear reprogramming in cultured ICM-derived pig cells after nuclear transfer has been reported (Ouhibi et al., 1996). The ability of the embryos to participate in normal development was not studied.
In a recent study in cattle, 9-13% of cleaved nuclear transplant embryos developed to the blastocyst stage when oogonia collected from female fetuses (50-70 days gestation) was utilized as nuclei donors (Lavoir et al., 1997). Although no live calves were produced, an abnormal conceptus developed in one animal that had received 4 embryos. This conceptus was recovered by induced abortion at day 43 after failing to detect a heartbeat, and genetic analysis showed the fetus to be genetically identical to the donor oogonia. Similar results using bovine PGCs from both male and female fetuses have been reported (Moens et al., 1996). The observation that nuclei from cultured bovine PGCs can direct development up to day 60 with no significant fetal abnormalities reported suggests that, when PGCs are placed in culture, nuclear changes occur that increase the nuclear potency of the cells when compared with freshly isolated PGCs (Strelchenko, 1996).
In spite of the foregoing reports, the technique of nuclear transfer is plagued by extremely low efficiency. Thus, methods and compositions that increase the efficiency of nuclear transfer, using both cultured somatic and germ cells, would represent a significant advance in the art.
SUMMARY OF THE INVENTION
The present invention overcomes one or more of the shortcomings in the art by providing methods and compositions using apoptosis inhibitors to increase the number of cells available for manipulation, including homologous recombination and gene targeting, in the generation of cell lines, transgenic and chimeric tissues and animals. The methods and compositions of the invention increase the efficiency of nuclear transfer, for use in the production of transgenic and non-transgenic mammals from cultured cells or cell lines, in cloning mammals, and for producing chimeric cell lines, transgenic and chimeric mammalian tissues and mammals.
The present invention thus provides methods and compositions for increasing, and preferably significantly increasing, the number and/or proportion of nuclear transfer-competent cells within a mammalian cell population. These methods and compositions comprise contacting, providing, administering, admixing or culturing a mammalian cell population with an amount of at least a first apoptosis inhibitor effective to increase, and preferably significantly increase, the number and/or proportion of nuclear transfer competent cells within the mammalian cell population.
The invention further provides methods and compositions for performing nuclear transfer in which the efficiency of nuclear transfer is increased, and preferably, significantly increased. Such methods and compositions comprise maintaining or culturing a mammalian cell population in the presence of an amount of at least a first apoptosis inhibitor effective to increase, and preferably significantly increase, the number and/or proportion of nuclear transfer competent cells within the cell population and fusing at least a first nuclear transfer-competent cell therefrom with a suitable enucleated, recipient cell or ovum.
A “nuclear transfer-competent cell”, as used herein, means a cell capable of being used in conjunction with a suitable enucleated, recipient cell in an effective nuclear transfer method. The present invention therefore provides methods and compositions for increasing, and preferably significantly increasing, the number and/or proportion of nuclear transfer-competent cells from within mammalian cell populations contemplated for use in nuclear transfer.
“Nuclear transfer-competent cells” are preferably in the G0/G1 stage of the cell cycle and, as used herein, are “viable” in the sense that they are capable of effectively participating in nuclear transfer in conjunction with suitable enucleated, recipient cell(s) to produce functional nucleated cells, reprogrammed nucleated cells, and reprogrammed nucleated cells capable of instructing the development of a new embryo. The invention thus provides methods and compositions for increasing, and preferably significantly increasing, the number and/or proportion of viable, nuclear transfer-competent cells (nuclear transfer-compeient, viable cells) from within cell populations contemplated for use in nuclear transfer.
In such methods and compositions of the invention, it is the contact, provision, administration, admixture or culture of the cell population comprising the cells for use in nuclear transfer in the presence of an effective amount of at least a first apoptosis inhibitor that increases or significantly increases the number and/or proportion of viable, nuclear transfer-competent cells.
To complete the nuclear transfer process, the viable, nuclear transfer-competent cell or cells is/are fused with suitable enucleated, recipient cell(s), thereby achieving nuclear transfer, i.e., transfer of the donor nucleus into the enucleated cell to produce a viable, nucleated cell, reprogrammed nucleated cell and/or reprogrammed, nucleated cell capable of instructing the development of a new embryo.
Accordingly, the invention provides increasingly effective and efficient methods and compositions for performing nuclear transfer. Such methods and compositions comprise culturing or maintaining a mammalian cell population containing at least some cells or a sub-population of cells at the G0/G1 stage of the cell cycle in media comprising an effective amount of at least a first apoptosis inhibitor, thereby increasing the number and/or proportion of viable cells within the G0/G1 stage cells of said cell population; and fusing at least a first viable G0/G1 cell with an enucleated mammalian ovum. The “effective amount” of the at least a first apoptosis inhibitor is an amount effective to increase the number and/or proportion of viable G0/G1 cells in the G0/G1 sub-population or overall cell population.
In the methods and compositions for performing nuclear transfer of the invention, the “mammalian cell population” is “at least a first mammalian cell population”, which at least a first mammalian cell population comprises at least one, some or a sub-population of cells at the G0/G1 stage of the cell cycle. Culture or maintenance with an effective amount of at least a first apo
Bazer Fuller
Lee Chang-Kyu
Piedrahita Jorge A.
Weaks Regina
Bracewell & Patterson LLP
Crouch Deborah
The Texas A&M University System
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