Successful cross-breeding of cloned pigs expressing endo-beta-galactosidase C and human decay accelerating factor.
BACKGROUND
For successful organ xenotransplantation, genetically engineered pigs have been actively produced. Our attention has focused on (i) reduction of alphaGal expression by its digestion enzyme, endo-beta-galactosidase C (EndoGalC), and (ii) inhibition of complement activation by human decay accelerating factor (hDAF). Cell sorting and nuclear transfer enabled the effective production of cloned pigs expressing transgene at high levels. We report the successful cross-breeding of pigs expressing EndoGalC and hDAF.


METHODS
After hDAF and EndoGalC genes were transfected into pig fibroblasts from the fetus of Landrace x Yorkshire and Meishan, respectively, transfected cells expressing transgenes effectively were collected using a cell sorter. Cloned pigs were produced using the technology of somatic cell nuclear transfer. After cross-breeding of cloned pigs, kidneys expressing both EndoGalC and hDAF were transplanted into baboons to examine the efficacy of gene transduction.


RESULTS
Well-designed cloned pigs were produced by cross-breeding. alphaGal expression levels in cloned pigs were reduced up to 2 to 14%, compared to that in wild-type pigs. hDAF expression reached about 10- to 70-fold, compared to that in human umbilical vein endothelial cells. No congenital deformity was observed. There was no problem of increased stillbirth rate or growth retardation. Hyperacute rejection could be avoided in such a cloned pig to baboon kidney transplantation without any treatment for anti-pig antibody removal. However, grafts suffered from fibrin deposition as early as 1 h after transplantation, and were rejected after 1 week.


CONCLUSIONS
Using a cell sorting system for effective collection of transfected cells, two types of cloned pigs were produced with a very high level of hDAF expression and a low level of alphaGal expression. Such genetic modification was effective in preventing hyperacute rejection, but there was an immediate lapse into procoagulation after transplantation, resulting in acute vascular rejection. Effective suppression of antibody binding to the graft would be necessary, even if a high level of hDAF is expressed.
Yazaki
Satoko
S
Prime Tech Ltd, Tsuchiura, Japan.
Iwamoto
Masaki
M
Onishi
Akira
A
Miwa
Yuko
Y
Suzuki
Shunichi
S
Fuchimoto
Dai-ichiro
D
Sembon
Shoichiro
S
Furusawa
Tadashi
T
Hashimoto
Michiko
M
Oishi
Takatsugu
T
Liu
DaGe
D
Nagasaka
Takaharu
T
Kuzuya
Takafumi
T
Maruyama
Shoichi
S
Ogawa
Haruko
H
Kadomatsu
Kenji
K
Uchida
Kazuharu
K
Nakao
Akimasa
A
Kobayashi
Takaaki
T
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D018958:CD55 Antigens / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D019976:Cloning, Organism; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006824:Hybridization, Genetic; D008297:Male; D053652:Nuclear Transfer Techniques; D010215:Papio; D010375:Pedigree; D034421:Sus scrofa; D019076:Transgenes; D014183:Transplantation, Heterologous; D000519:alpha-Galactosidase / Q000235:genetics / Q000378:metabolism
D018958:CD55 Antigens; D006026:Glycoside Hydrolases; C057030:endo-beta-D-galactosidase C; D000519:alpha-Galactosidase