Complete oocyte activation using an oocyte-modifying agent and a reducing agent

A method for transplanting a nucleus from a mammalian cell to a recipient mammalian oocyte, comprising:

• (a) isolating the membrane-bounded nucleus from said mammalian cell;
• (b) removing the nuclear chromosomal material from said recipient mammalian oocyte, thereby preparing an enucleated recipient mammalian oocyte;
• (c) introducing said membrane-bounded nucleus from said mammalian cell into said enucleated recipient mammalian oocyte to form an oocyte containing said nucleus from said mammalian cell;
• (d) reprogramming the developmental cascade of events of said nucleus from said mammalian cell;
• (e) incubating said oocyte containing said nucleus from said mammalian cell of step (d) with an oocyte-modifying agent followed by a reducing agent, wherein said oocyte is incubating with said oocyte-modifying agent and said reducing agent, respectively, for a time and under conditions such that said oocyte is activated;
• and
• (f) culturing said activated oocyte of step (e) in vitro or in vivo.

A method for transplanting a nucleus from a mammalian embryo to a recipient mammalian oocyte, comprising:

• (a) isolating the membrane-bounded nucleus from a cell of said mammalian embryo;
• (b) removing the nuclear chromosomal material from said recipient mammalian oocyte, thereby preparing an enucleated recipient mammalian oocyte;
• (c) introducing said membrane-bounded nucleus from said cell of said mammalian embryo into said enucleated recipient mammalian oocyte to form an oocyte containing said nucleus from said cell of said mammalian embryo;
• (d) reprogramming the developmental cascade of events of said nucleus from said cell of said mammalian embryo;
• (e) incubating said oocyte containing said nucleus from said cell of said mammalian embryo of step (d) with an oocyte-modifying agent followed by a reducing agent, wherein said oocyte is incubating with said oocyte-modifying agent and said reducing agent, respectively, for a time and under conditions such that said oocyte is activated;
• and
• (f) culturing said activated oocyte of step (e) in vitro or in vivo.

A method for transplanting a nucleus from a mammalian fetus to a recipient mammalian oocyte, comprising:

• (a) isolating the membrane-bounded nucleus from a cell of said mammalian fetus;
• (b) removing the nuclear chromosomal material from said recipient mammalian oocyte, thereby preparing an enucleated recipient mammalian oocyte;
• (c) introducing said membrane-bounded nucleus from said cell of said mammalian fetus into said enucleated recipient mammalian oocyte to form an oocyte containing said nucleus from said cell of said mammalian fetus;
• (d) reprogramming the developmental cascade of events of said nucleus from said cell of said mammalian fetus;
• (e) incubating said oocyte containing said nucleus from said cell of said mammalian fetus of step (d) with an oocyte-modifying agent followed by a reducing agent, wherein said oocyte is incubating with said oocyte-modifying agent and said reducing agent, respectively, for a time and under conditions such that said oocyte is activated;
• and
• (f) culturing said activated oocyte of step (e) in vitro or in vivo.

A method for transplanting a nucleus from an adult mammal to a recipient mammalian oocyte, comprising:

• (a) isolating the membrane-bounded nucleus from a cell of said adult mammal;
• (b) removing the nuclear chromosomal material from said recipient mammalian oocyte, thereby preparing an enucleated recipient mammalian oocyte;
• (c) introducing said membrane-bounded nucleus from said cell of said adult mammal into said enucleated recipient mammalian oocyte to form an oocyte containing said nucleus from said cell of said adult mammal;
• (d) reprogramming the developmental cascade of events of said nucleus from said cell of said adult mammal;
• (e) incubating said oocyte containing said nucleus from said cell of said adult mammal of step (d) with an oocyte-modifying agent followed by a reducing agent, wherein said oocyte is incubating with said oocyte-modifying agent and said reducing agent, respectively, for a time and under conditions such that said oocyte is activated;
• and
• (f) culturing said activated oocyte of step (e) in vitro or in vivo.

A method for producing a cloned mammalian embryo, comprising:

• (a) isolating a membrane-bounded nucleus from a cell of said mammalian embryo;
• (b) removing the nuclear chromosomal material from a recipient mammalian oocyte, thereby preparing an enucleated recipient mammalian oocyte;
• (c) introducing said membrane-bounded nucleus from said cell of said mammalian embryo into said enucleated recipient mammalian oocyte to form an oocyte containing said nucleus from said cell of said mammalian embryo;
• (d) reprogramming the developmental cascade of events of said nucleus from said cell of said mammalian embryo;
• (e) incubating said oocyte containing within its cytoplasm said nucleus from said cell of said mammalian embryo of step (d) with an oocyte-modifying agent followed by a reducing agent, wherein said oocyte is incubating with said oocyte-modifying agent and said reducing agent, respectively, for a time and under conditions such that said oocyte is activated;
• and
• (f) culturing said activated oocyte of step (e) in vivo or in vitro to produce a cloned mammalian embryo.

A method for producing a cloned mammal, comprising:

• (a) isolating a membrane-bounded nucleus from a cell of said mammal;
• (b) removing the nuclear chromosomal material from an unfertilized recipient mammalian oocyte, thereby preparing an enucleated recipient mammalian oocyte;
• (c) introducing said membrane-bounded nucleus from said cell of said mammal into said enucleated recipient mammalian oocyte to form an oocyte containing said nucleus from said cell of said mammal;
• (d) reprogramming the developmental cascade of events of said nucleus from said cell of said mammal;
• (e) incubating said oocyte containing within its cytoplasm said nucleus of said cell from said mammal with an oocyte-modifying agent followed by a reducing agent, wherein said oocyte is incubating with said oocyte-modifying agent and said reducing agent, respectively, for a time and under conditions such that said oocyte is activated;
• (f) culturing said activated oocyte of step (e) in vitro or in vivo;
• and
• (g) transferring said cultured, activated oocyte of step (f) to the oviduct or uterus of a recipient maternal mammal to produce a cloned mammal.

A method of studying genetic imprinting in a mammal, comprising:

• (a) incubating an unfertilized mammalian oocyte with an oocyte-modifying agent followed by a reducing agent,
• wherein said unfertilized mammalian oocyte is incubating with said oocyte-modifying agent and said reducing agent, respectively, for a time and under conditions such that said unfertilized mammalian oocyte is activated;
• and
• (b) determining whether a gene of interest is expressed or not during culturing of said activated mammalian oocyte.