Transfection, storage and transfer of male germ cells for generation of selectable transgenic stem cells

A method of obtaining a selectable transgenic stem cell of a vertebrate, comprising:

• administering to a gonad of a male vertebrate a transfection mixture comprising at least one transfecting agent and at least one polynucleotide comprising a transcriptional unit of a cyclin A1 promoter sequence operatively linked to a DNA encoding a fluorescent or light-emitting protein, under conditions effective to reach a germ cell or germ cell precursor of the male vertebrate;
• and
• causing said polynucleotide to be taken up by, and released into, said germ cell or precursor cell;
• incorporating said polynucleotide into the genome of said germ cell or precursor cell, whereby a selectable transgenic stem cell is obtained expressing said fluorescent or light-emitting protein, by which said stem cell can be isolated or selected from a non-stem cell.

A method of obtaining a selectable transgenic stem cell of a vertebrate, comprising:

• administering to a gonad of a male vertebrate a transfection mixture comprising at least one transfecting agent and at least one polynucleotide comprising a transcriptional unit of a cyclin A1 promoter sequence operatively linked to a DNA encoding a fluorescent or light-emitting protein, under conditions effective to reach a germ cell or germ cell precursor of the male vertebrate;
• and
• causing said polynucleotide to be taken up by, and released into, said germ cell or precursor cell;
• incorporating said polynucleotide into the genome of said germ cell or precursor cell;
• allowing said germ cell or precursor cell to develop into a maturing male gamete;
• and
• breeding said male vertebrate with a female of its species to obtain a transgenic progeny expressing said fluorescent or light-emitting protein in at least one of its stem cells, whereby said stem cell can be isolated or selected from a non-stem cell.

A method of obtaining a selectable stem cell, comprising:

• obtaining a maturing male germ cell from a vertebrate;
• transfecting said male germ cell in vitro with at least one polynucleotide comprising a transcriptional unit of a stem cell-specific promoter operatively linked to a DNA encoding a fluorescent or light-emitting protein, in the presence of a gene delivery mixture comprising at least one transfecting agent, at about or below the vertebrate's body temperature and for a transfection-effective period of time;
• causing said polynucleotide to be taken up by, and released into said germ cell;
• and fertilizing an ovum with said germ cell such that a transgenic progeny expressing said fluorescent or light-emitting protein in at least one of its stem cells is obtained, said stem cell(s) being selectable from non-stem cells by detecting light emissions from said stem cell(s).

A method of obtaining a selectable stem cell, comprising:

• obtaining a maturing male germ cell from a vertebrate;
• transfecting said male germ cell in vitro with at least one polynucleotide comprising a transcriptional unit of a cyclin A1 promoter operatively linked to a DNA encoding a fluorescent or light-emitting protein, in the presence of a gene delivery mixture comprising at least one transfecting agent, at about or below the vertebrate's body temperature and for a transfection-effective period of time;
• and
• allowing said polynucleotide to be taken up by, and released into said germ cell;
• fertilizing an ovum with said germ cell such that a transgenic progeny expressing said fluorescent or light-emitting protein in at least one of its stem cells is obtained, said stem cell(s) being selectable from non-stem cells by detecting light emissions from said stem cell(s).