Genetic modification of primordial germ cells by gene trapping, gene targeting, and phiC31 integrase.
The genome of germline committed cells is thought to be protected by mechanisms of transcriptional silencing, posing a barrier to transgenesis using cultured germline cells. We found that selection for transgene integration into the primordial germ cell genome required that the transgenes be flanked by the chicken beta-globin insulator. However, integration frequency was low, and sequencing of the insertion sites revealed that the transgenes preferentially inserted into active promoter regions, implying that silencing prohibited recovery of insertions in other regions. Much higher frequencies of integration were achieved when the phiC31 integrase was used to insert transgenes into endogenous pseudo attP sites. Despite the evidence for transcriptional silencing in PGCs, gene targeting of a nonexpressed gene was also achieved. The ability to make genetic modifications in PGCs provides unprecedented opportunities to study the biology of PGCs, as well as produce transgenic chickens for applications in biotechnology and developmental biology.
Leighton
Philip A
PA
Origen Therapeutics, 1450 Rollins Road, Burlingame, California 94010, USA. pleight@origentherapeutics.com
van de Lavoir
Marie-Cecile
MC
Diamond
Jennifer H
JH
Xia
Chunyao
C
Etches
Robert J
RJ
D000818:Animals; D001483:Base Sequence; D002642:Chick Embryo / Q000502:physiology; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017931:DNA Primers; D004270:DNA, Circular / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005854:Germ Cells / Q000201:enzymology / Q000502:physiology*; D019426:Integrases / Q000235:genetics*; D008969:Molecular Sequence Data
D017931:DNA Primers; D004270:DNA, Circular; D019426:Integrases