Sequential targeting of the genes encoding immunoglobulin-mu and prion protein in cattle.
Gene targeting is accomplished using embryonic stem cells in the mouse but has been successful, only using primary somatic cells followed by embryonic cloning, in other species. Gene targeting in somatic cells versus embryonic stem cells is a challenge; consequently, there are few reported successes and none include the targeting of transcriptionally silent genes or double targeting to produce homozygotes. Here, we report a sequential gene targeting system for primary fibroblast cells that we used to knock out both alleles of a silent gene, the bovine gene encoding immunoglobulin-mu (IGHM), and produce both heterozygous and homozygous knockout calves. We also carried out sequential knockout targeting of both alleles of a gene that is active in fibroblasts, encoding the bovine prion protein (PRNP), in the same genetic line to produce doubly homozygous knockout fetuses. The sequential gene targeting system we used alleviates the need for germline transmission for complex genetic modifications and should be broadly applicable to gene functional analysis and to biomedical and agricultural applications.
Kuroiwa
Yoshimi
Y
Gemini Science, 3030 Bunker Hill Street #226, San Diego, California 92109, USA.
Kasinathan
Poothappillai
P
Matsushita
Hiroaki
H
Sathiyaselan
Janaki
J
Sullivan
Eddie J
EJ
Kakitani
Makoto
M
Tomizuka
Kazuma
K
Ishida
Isao
I
Robl
James M
JM
D000483:Alleles; D000818:Animals; D002417:Cattle; D018390:Gene Targeting; D007148:Immunoglobulin mu-Chains / Q000235:genetics*; D011328:Prions / Q000235:genetics*
D007148:Immunoglobulin mu-Chains; D011328:Prions