本帖最后由 SCNT 于 2011-5-22 15:33 编辑 3 [8 y, S$ F5 x) Q2 y; g! N . q* O" G& Q' G3 d i2 A! GBlastocysts derived from adult fibroblasts of a rhesus monkey 1 E- L1 P$ W% v& t(Macaca mulatta) using interspecies somatic cell nuclear transfer : k% J6 m$ \4 e7 _: p6 s5 U# ?* H1 w9 S- \
Dae Kee Kwon2, Jung Taek Kang2, Sol Ji Park2, Ma Ninia Limas Gomez2, Su Jin Kim,2,7 K& ?# x* ?8 @- U/ e# z
Mohammad Atikuzzaman2, Ok Jae Koo2, Goo Jang2 and Byeong Chun Lee1 , [0 d, S; ~3 I6 w& }Department of Theriogenology and Biotechnology, College of Veterinary Medicine and Research Institute for Veterinary2 o6 p; l- ]3 o% g$ S
Science, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151–742, Republic of Korea2 i' E& C% T3 B& `& L$ q7 {
% o3 K" C; ?: _& z5 B: ASummary% a# \6 h6 F5 Y5 W
In non-human primates, it is difficult to collect sufficient numbers of oocytes for producing identical6 u3 p" {$ K2 y* Q% ?4 P
embryos by somatic cell nuclear transfer (SCNT). Because of this factor, inter-species SCNT (iSCNT)6 J5 f" B. _6 K" i, E7 \
using heterospecific oocytes is an attractive alternative approach. The objective of this study was to# Q5 [! H# W+ o; j, H; D
produce iSCNT-derived blastocysts using enucleated cow (Bos taurus) metaphase II oocytes and adult 6 t$ {! |$ V' K5 Trhesus monkey (Macaca mulatta) fibroblasts. Ear skin tissue from a 6-year-old male rhesus monkey! v6 g. p2 _/ M0 k8 b8 x
was collected by biopsy and fibroblasts were isolated. Immature cumulus–oocyte complexes from cow) K; H4 v5 q0 u% Z) j9 G; b
ovaries were collected and matured in vitro in Medium 199. The enucleated oocytes were reconstructed 7 m9 ^3 n% O1 f) x D! g9 w5 ^with rhesus monkey fibroblasts and iSCNT embryos were cultured in modified synthetic oviduct fluid2 T }9 Y# h" Y# _8 V
in an atmosphere of 5–5.5% CO2 under various conditions (37–39 ◦C and 5–20% O2) to examine the * L m) D/ m9 m, X; \% zeffects of in vitro culture conditions. Most embryos were arrested at the 8- or 16-cell stage and only7 E3 J2 {' y) z$ v( O
three blastocysts were derived in this way using iSCNT from a total of 1153 cultured activated embryos' I% j" a: m( u T, Y* C3 x& u, `
(0.26% production rate). Two of the three blastocysts were used for counting nuclear numbers using$ b) U4 h) x# k: x! p
bisbenzimide staining, which were 51 and 24. The other iSCNT-derived blastocyst was used to analyse 1 H+ X( A" z- c* r( dmitochondrial DNA (mtDNA) by PCR, and both rhesus monkey and cow mtDNA were detected. 3 ?% E( S& w, `- X0 x, v& JAlthough the development rate was extremely low, this study established that iSCNT using two 4 @, l" ]6 g* u0 f7 J' d0 G; cphylogenetically distant species, including a primate, could produce blastocysts. With improvements6 _0 d8 ^4 h' T8 {6 F Q
in the development rate, it may be possible to produce rhesus monkey iSCNT-derived embryonic stem 0 j4 H+ h! k- b+ i2 Scell lines for studies on primate nucleus and cow mitochondria interaction mechanisms.3 ^/ i0 _, i: ^+ V: a
摘要：非人灵长类很难收集到足够的卵母细胞用于生产SCNT胚胎。由于这个因素的影响，使用异种卵母细胞进行异种SCNT是一种非常吸引人的可供选择的方法。本研究的目的是使用去核牛卵母细胞和猕猴成年成纤维细胞生产异种SCNT囊胚。收集6岁猕猴耳组织块，分离成纤维细胞。收集牛卵巢内的COCS进行体外成熟培养。去核牛卵母细胞与猕猴成纤维细胞构建异种SCNT胚胎，转入改良输卵管合成液中，培养于5-5.5%CO2、37-39度、5-20%O2条件下检测不同培养条件对胚胎发育的影响。大部分胚胎阻止于8-细胞或16-细胞阶段，共计1153枚胚胎仅获得3枚囊胚。取2个囊胚进行染色记数囊胚细胞数，分别是51和24。另外一个囊胚通过PCR分析mtDNA，以确认猕猴和牛mtDNA。尽管囊胚发育率极低，但本研究表明，使用2个远距离物种进行异种SCNT可以获得囊胚。为分离猕猴异种SCNT干细胞系提供可能，进而可以研究灵长类核和牛线粒体互作机制。