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Genetic modification of primordial germ cells by gene trapping, gene targeting, and ϕC31 integrase
Author(s) -
Leighton Philip A.,
van de Lavoir MarieCecile,
Diamond Jennifer H.,
Xia Chunyao,
Etches Robert J.
Publication year - 2008
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.20859
Subject(s) - biology , germline , transgene , genetics , gene silencing , gene , transgenesis , gene targeting , integrase , genome , reproductive technology , embryogenesis
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 β‐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 ϕC31 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. Mol. Reprod. Dev. 75: 1163–1175, 2008. © 2008 Wiley‐Liss, Inc.