Nuclear transfer using cells cultured in serum starvation media containing apoptosis inhibitors

A method of growing fetal or embryonic fibroblasts or primordial germ cells, comprising growing a cell culture comprising fetal or embryonic fibroblasts or primordial germ cells on an effective density of feeder cells and in a biologically effective serum starvation medium comprising an amount of at least a first apoptosis inhibitor effective to increase the number of cells at the G0/G1 stage of the cell cycle when said cell culture is maintained for a time sufficient to obtain undifferentiated fetal or embryonic fibroblasts or primordial germ cells.

A method of nuclear transfer, comprising culturing a mammalian cell population containing cells at the G0/G1 stage of the cell cycle in serum starvation media comprising an amount of at least a first apopotosis inhibitor effective to increase the proportion of viable G0/G1 cells in said cell population;

• and fusing at least a first viable G0/G1 cells with an enucleated mammalian ovum.

A method of preparing mammalian cells at the G0/G1 stage of the cell cycle that contain a selected DNA segment, comprising:

• a) culturing a mammalian cell population containing cells at the G0/G1 stage of the cell cycle in serum starvation media comprising an amount of at least a first apoptosis inhibitor effective to increase the proportion of viable G0/G1 cells in said cell population;
• and
• b) introducing said DNA segment into the viable G0/G1 cells in said cell population.

A method of nuclear transfer, comprising culturing a cell population containing bovine or porcine fetal or embryonic fibroblasts or primordial germ cells at the G0/G1 stage of the cell cycle in serum starvation media comprising an amount of at least a first apoptosis inhibitor effective to increase the proportion of viable G0/G1 cells in said cell population;

• and fusing at least a first viable G0/G1 cell from the cultured cell population with an enucleated bovine or porcine ovum, thereby achieving nuclear transfer.

A method of nuclear transfer, comprising culturing a mammalian cell population containing cells at the G0/G1 stage of the cell cycle in serum starvation media comprising an amount of the apoptosis inhibitor α2-macroglobulin (MAC) effective to increase the proportion of viable G0/G1 cells in said cell population when present substantially throughout the culture of said cell population;

• and fusing at least a first viable G0/G1 cell from the cultured cell population with an enucleated mammalian ovum, thereby achieving nuclear transfer.

A method of nuclear transfer, comprising culturing a mammalian cell population containing cells at the G0/G1 stage of the cell cycle in serum starvation media comprising an apoptosis inhibitor selected from the group consisting of N-acetylcysteine (NAC), butylated hydroxyanisole (BHA), cimetidine (CIM), N-t-butyl-α-phenylnitrone (BPN) and glutathione (GSH) in an amount effective to increase the proportion of viable G0/G1 cells in said cell population;

• and fusing at least a first viable G0/G1 cell from the cultured cell population with an enucleated mammalian ovum, thereby achieving nuclear transfer.

A method of nuclear transfer, comprising culturing a mammalian cell population containing cells at the G0/G1 stage of the cell cycle in serum starvation media comprising an amount of the apoptosis inhibitor N-acetylcysteine (NAC) effective to increase the proportion of viable G0/G1 cells in said cell population when present at the initial stages of the culture of said cell population;

• and fusing at least a first viable G0/G1 cell from the cultured cell population with an enucleated mammalian ovum, thereby achieving nuclear transfer.

A method of nuclear transfer, comprising culturing a mammalian cell population containing cells at the G0/G1 stage of the cell cycle in serum starvation media comprising at least a first serine protease apoptosis inhibitor and at least a second antioxidant apoptosis inhibitor in an amount effective to increase the proportion of viable G0/G1 cells in said cell population;

• and fusing at least a first viable G0/G1 cell from the cultured cell population with an enucleated mammalian ovum, thereby achieving nuclear transfer.

A method of nuclear transfer, comprising culturing a mammalian cell population containing cells at the G0/G1 stage of the cell cycle in serum starvation media comprising an amount of at least a first serine protease apoptosis inhibitor other than a2-macroglobulin effective to increase the proportion of viable G0/G1 cells in said cell population;

• and fusing at least a first viable G0/G1 cell with an enucleated mammalian ovum.

A method of nuclear transfer, comprising culturing a mammalian cell population containing cells at the G0/G1 stage of the cell cycle in serum starvation media comprising an amount of at least a first antioxidant apoptosis inhibitor other than thioredoxin in an amount effective to increase the proportion of viable G0/G1 cells in said cell population;

• and fusing at least a first viable G0/G1 cell with an enucleated mammalian ovum.

A method of nuclear transfer, comprising culturing a mammalian cell population containing cells at the G0/G1 stage of the cell cycle in serum starvation media comprising an amount of at least a first apoptosis inhibitor other than α2-macroglobulin, thioredoxin or uteroferrin in an amount effective to increase the proportion of viable G0/G1 cells in said cell population;

• and fusing at least a first viable G0/G1 cell with an enucleated mammalian ovum.

A method of cloning a mammal from somatic or primordial germ cells, comprising:

• (a) culturing a population of mammalian somatic or primordial germ cells containing cells at the G0/G1 stage of the cell cycle in serum starvation media comprising an effective amount of at least a first apoptosis inhibitor for a period of time suitable to increase the proportion of viable G0/G1 cells in said population;
• and
• (b) generating a viable cloned mammal from at least a first of said viable G0/G1 cells.