Production of alpha-1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations.
Hyperacute rejection of porcine organs by old world primate recipients is mediated through preformed antibodies against galactosyl-alpha-1,3-galactose (Galalpha-1,3-Gal) epitopes expressed on the pig cell surface. Previously, we generated inbred miniature swine with a null allele of the alpha-1,3-galactosyltransferase locus (GGTA1) by nuclear transfer (NT) with gene-targeted fibroblasts. To expedite the generation of GGTA1 null pigs, we selected spontaneous null mutant cells from fibroblast cultures of heterozygous animals for use in another round of NT. An unexpectedly high rate of spontaneous loss of GGTA1 function was observed, with the vast majority of null cells resulting from loss of the WT allele. Healthy piglets, hemizygous and homozygous for the gene-targeted allele, were produced by NT by using fibroblasts that had undergone deletional and crossover/gene conversion events, respectively. Aside from loss of Galalpha-1,3-Gal epitopes, there were no obvious phenotypic differences between these null piglets and WT piglets from the same inbred lines. In fact, congenital abnormalities observed in the heterozygous NT animals did not reappear in the serially produced null animals.
Kolber-Simonds
Donna
D
Immerge Biotherapeutics, Inc., 300 Technology Square, Cambridge, MA 02139, USA.
Lai
Liangxue
L
Watt
Steven R
SR
Denaro
Maria
M
Arn
Scott
S
Augenstein
Monica L
ML
Betthauser
Jeffery
J
Carter
David B
DB
Greenstein
Julia L
JL
Hao
Yanhong
Y
Im
Gi-Sun
GS
Liu
Zhonghua
Z
Mell
Greg D
GD
Murphy
Clifton N
CN
Park
Kwang-Wook
KW
Rieke
August
A
Ryan
David J J
DJ
Sachs
David H
DH
Forsberg
Erik J
EJ
Prather
Randall S
RS
Hawley
Robert J
RJ
D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D005347:Fibroblasts / Q000648:ultrastructure; D005434:Flow Cytometry; D005700:Galactosyltransferases / Q000235:genetics*; D019656:Loss of Heterozygosity; D053652:Nuclear Transfer Techniques; D010641:Phenotype; D013552:Swine
D005700:Galactosyltransferases