title: Improvement of cloning efficiency in minipigs using post-thawed donor cells treated with roscovitine.
abstract: Massachusetts General Hospital miniature pigs (MGH minipigs) have been established for organ transplantation studies across the homozygous major histocompatibility complex, but cloning efficiency of MGH minipigs is extremely low. This study was designed to increase the productivity of MGH minipigs by nuclear transfer of post-thaw donor cells after 1 h co-incubation with roscovitine. The MGH minipig cells were genetically modified with GT KO (alpha1,3-galactosyltransferase knock-out) and hCD46 KI (human CD46 knock-in) and used as donor cells. The GT KO/hCD46 KI donor cells were cultured for either 3 days (control group) or 1 h after thawing with 15 μM roscovitine (experimental group) prior to the nuclear transfer. The relative percentage of the transgenic donor cells that entered into G0/G1 was 93.7 % (±2.54). This was different from the donor cells cultured for 1 h with the roscovitine-treated group (84.6 % ±4.6) (P < 0.05) and without roscovitine (78.6 % ±5.5) (P < 0.01), respectively. The pregnancy rate and delivery rate in the roscovitine group (8/12 and 6/8, respectively) were significantly higher (P < 0.01) than those in the control group (6/19 and 3/6, respectively). In the experimental group, 12 GT KO/hCD46 KI transgenic minipigs were successfully generated, and five minipigs among them survived for more than 6 months so far. The recipient-based individual cloning efficiency ranged from 0.74 to 2.54 %. In conclusion, gene-modified donor cells can be used for cloning of MGH minipigs if the cells are post-thawed and treated with roscovitine for 1 h prior to nuclear transfer.
authors.affiliation: Animal Biotechnology Division, National Institute of Animal Science, RDA, Gyeonggi-do, 441-706, Republic of Korea, hwangss@korea.kr.
authors.forename: Seongsoo
authors.initials: S
authors.lastname: Hwang
authors.forename: Keon Bong
authors.initials: KB
authors.lastname: Oh
authors.forename: Dae-Jin
authors.initials: DJ
authors.lastname: Kwon
authors.forename: Sun-A
authors.initials: SA
authors.lastname: Ock
authors.forename: Jeong-Woong
authors.initials: JW
authors.lastname: Lee
authors.forename: Gi-Sun
authors.initials: GS
authors.lastname: Im
authors.forename: Sung-Soo
authors.initials: SS
authors.lastname: Lee
authors.forename: Kichoon
authors.initials: K
authors.lastname: Lee
authors.forename: Jin-Ki
authors.initials: JK
authors.lastname: Park
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000379:methods*; D005260:Female; D005700:Galactosyltransferases / Q000235:genetics; D055879:Gene Knock-In Techniques; D055786:Gene Knockout Techniques; D006801:Humans; D051916:Membrane Cofactor Protein / Q000235:genetics; D053652:Nuclear Transfer Techniques; D010053:Ovary / Q000187:drug effects* / Q000378:metabolism; D011247:Pregnancy; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011687:Purines / Q000494:pharmacology*; D000077546:Roscovitine; D013552:Swine; D013556:Swine, Miniature / Q000196:embryology / Q000235:genetics* / Q000254:growth & development
chemical_list: C497857:CD46 protein, human; D051916:Membrane Cofactor Protein; D047428:Protein Kinase Inhibitors; D011687:Purines; D000077546:Roscovitine; D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase