Germline transmission of avian primordial germ cells (PGCs)
The present invention is long-term cultures of avian PGCs and techniques to produce germline chimeric and transgenic birds derived from prolonged PGC cultures. In some embodiments, these PGCs can be transfected with genetic constructs to modify the DNA of the PGC, specifically to introduce a transgene encoding an exogenous protein. When combined with a host avian embryo by known procedures, those modified PGCs are transmitted through the germline to yield transgenic offspring. These germline chimeric birds do not have substantial contributions of PGC-derived phenotypes in somatic cells or tissues. This invention includes compositions comprising long-term cultures of PGCs that can be genetically modified by gene targeting, that can accept large amounts of foreign DNA and that contribute to the germline of recipient embryos.
1 . A clonally-derived cell culture of chicken primordial germ cells carrying exogenous DNA stably integrated genome of the primordial germ cells.
2 . The culture of claim 1 wherein the culture medium is comprised of buffalo rat liver (BRL) cells.
3 . The culture of claim 1 wherein the culture medium is comprised of fibroblast growth factor (FGF).
4 . The culture of claim 1 wherein the culture medium is comprised of stem cell factor.
5 . The culture of claim 1 wherein the culture medium is comprised of chicken serum.
6 . The culture of claim 1 wherein the culture comprises at least 1×10 5 cells.
7 . The culture of claim 1 wherein the exogenous DNA is flanked by at least one insulator.
8 . A germline chimeric chicken comprising:
germline tissues colonized by genetically modified primordial germ cells, and
somatic tissues substantially free of genetically modified cells.
9 . The germline chimera of claim 8 wherein the genetic modification is the stable integration of exogenous DNA.
10 . The germline chimera of claim 9 wherein the exogenous DNA encodes a protein.
11 . The germline chimera of claim 10 wherein protein is a monoclonal antibody.
12 . The method of claim 11 wherein the monoclonal antibody has a human polynucleotide sequence.
13 . The germline chimera of claim 10 wherein the exogenous DNA is flanked by at least one insulator.
14 . A primordial germ cell comprising a stably integrated transgene having an expressable early response to neural induction (ERNI) promoter operatively linked to a coding region of the transgene.