本帖最后由 SCNT 于 2011-5-23 13:49 编辑 / {+ x. n, |5 ~2 v+ W/ ^5 ^- C' U( B! r# L# e* \
Production of transgenic canine embryos using interspecies somatic7 l/ k" a" o( N: k" ~% O# O
cell nuclear transfer * i' G; W* c* @5 _So Gun Hong2, Hyun Ju Oh2, Jung Eun Park2, Min Jung Kim2, Geon A. Kim2, Ok Jae Koo2, ! t! h b5 t( @8 t* C+ o3 GGoo Jang2 and Byeong Chun Lee16 F) v9 u* h; Q# y$ a1 b% a; k; W
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Gwanak-gu,; X/ B/ H2 x+ z/ w9 |5 P9 E# y( e
Seoul, Korea : |, m7 i: h7 L @2 g! ]9 IDate submitted: 10.03.2010. Date accepted: 29.09.2010 ( i3 N6 ^3 Y0 h% m0 n/ ~Summary; s& n4 w1 W3 I, Z% W
Somatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals9 o! n( l6 N+ O2 |! Y
and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured & \( K4 x/ }4 w( D5 aoocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells2 m( n5 c' t4 g
carry ‘foreign’ DNA and are clones when all the donor cells are genetically identical. However, in) _$ l$ _3 w0 s8 J4 M% x8 ~
canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency / _ m& G2 ?' c. q/ [3 Xof in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is * F6 Z, g7 f/ J0 ^$ nto use oocytes from a different species or even a different genus, such as bovine oocytes, that can be$ n! a) g( s5 B( A% C; W
matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast- N% B- J- u, i# _2 u
line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic% `, P" F6 \# d) n3 i* O/ [( y
development of canine cloned embryos derived from transgenic and non-transgenic cell lines using, e {$ U5 O3 w
bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the " f" v: W; M* G% L8 R3 ^GFP and puromycin resistance genes using FuGENE 6 R . Viability levels of these cells were determined ; P+ A& ~/ f6 v) Hby the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT 8 P- [) X3 y& U5 u(iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT9 ?, i# D& Z. E/ \. A
measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different9 u5 }* `. \( c8 O! D' q
from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic " [- @# x6 t5 E5 jiSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), 9 s; H1 v! O6 {. A) F& [6 k1 J0 zcleavage rates (69.7% vs. 73.8%) and development to the 8–16-cell stage (40.1% vs. 42.7%). Embryos" f$ F; R: V2 G0 ]5 ?
derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8–16-cell stages 2 h4 i0 i R3 m: b* x( i2 }3 Zwithout mosaicism. In summary, our results demonstrated that, following successful isolation of canine 5 {# M* D+ V$ ` k4 @2 ~- _# R- P9 Gtransgenic cells, iSCNT embryos developed to early pre-implantation stages in vitro, showing stable 8 @: o9 s9 }8 R4 X3 M& ~3 XGFP expression. These canine–bovine iSCNT embryos can be used for further in vitro analysis of canine% Q, Q% I; L6 I( R
transgenic cells and will contribute to the production of various transgenic dogs for use as specific/ c: N# s$ J0 _3 A! N: n7 C
human disease models.; ?% L" I4 `5 i1 k% D+ ^7 a
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摘要：SCNT已经成为生产转基因动物的重要工具。然而，对于狗，由于卵母细胞体外成熟的效率非常低下，很难获得足够的成熟卵母细胞用于SCNT，阻碍了转基因克隆狗的发展。其中一个方案是使用其它物种的卵母细胞，比如牛卵母细胞，可以很容易进行体外成熟。因此，本研究的目的是（1）建立表达GFP的狗胎儿成纤维细胞系；（2）使用牛卵母细胞做受体，转基因或非转基因细胞做供体，研究克隆狗胚胎的体外发育。狗胎儿成纤维细胞使用FuGENE 6 R 试剂盒转染包含GFP和抗嘌呤霉素基因的质粒。细胞的发育能力通过MTT进行检测。转基因和非转基因细胞MTT检测没有显著差异。转基因和非转基因异种胚胎在融合率（73.1% and 75.7%）、分裂率以及8-16细胞阶段效率均没有差异。来自转基因细胞的胚胎，在2-细胞、4-细胞和8-16细胞阶段完全表达GFP。结论，异种胚胎可以在体外发育，并表达GFP。牛-狗异种胚胎可以用于狗转基因细胞的体外分析，有助于生产不同的转基因狗，可以作为人类疾病模型。 " E9 d9 t/ M6 I: m) f