[原创]2011英文翻译使用异种SCNT技术生产转基因狗胚胎

SCNT

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Production of transgenic canine embryos using interspecies somatic- m5 e% q  u( _- W# b
cell nuclear transfer
- e5 A. m2 A' G4 w" ]8 F# _So Gun Hong2, Hyun Ju Oh2, Jung Eun Park2, Min Jung Kim2, Geon A. Kim2, Ok Jae Koo2,
( a; R# O$ D! a* ~1 M) gGoo Jang2 and Byeong Chun Lee1- @/ K2 ^& S: f5 Y8 U! Q- D
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Gwanak-gu,( E- q. U9 x2 S$ S5 K) r& r" p, y
Seoul, Korea& ]+ W; E+ y9 |
Date submitted: 10.03.2010. Date accepted: 29.09.2010
2 \' d- s5 `  j  S2 d* d& D5 FSummary% \- U3 s- r9 N/ W& J" U+ H
Somatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals" m9 k: g6 r, d) D" H6 d* A% F
and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured
+ e* l) p9 \  ^2 U; T$ F2 b! Xoocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells
. P7 q0 Z4 R& J# ]7 ^' ]" vcarry ‘foreign’ DNA and are clones when all the donor cells are genetically identical. However, in
: @! `; l4 p6 W+ ~canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency/ F; `0 [! E& k
of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is
# _' M9 E2 F+ j7 p1 n1 tto use oocytes from a different species or even a different genus, such as bovine oocytes, that can be
% E8 K- y1 u% Y  B# ]! N7 p3 bmatured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast: l* w$ J) L! T) B! o
line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic
& i: d+ H, Y6 j- ?development of canine cloned embryos derived from transgenic and non-transgenic cell lines using
' m" [5 X4 S5 }; d9 zbovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the; i7 t* H0 H$ \4 Z& N
GFP and puromycin resistance genes using FuGENE 6 R  . Viability levels of these cells were determined
' `5 l2 u* n& M/ oby the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT
+ T1 q$ q1 _2 T3 `! A8 p(iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT
* S, e' a/ @8 r$ b6 ~' C* qmeasurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different
: ^. `  t" _4 ffrom non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic
2 z% h' W/ N" }& a4 ^, }iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively),
8 t5 d* |  k/ O) o, U; \cleavage rates (69.7% vs. 73.8%) and development to the 8–16-cell stage (40.1% vs. 42.7%). Embryos
  |$ `: s# E+ q3 q& @' N2 m1 [, Pderived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8–16-cell stages$ X0 [2 C; X  @) f, v
without mosaicism. In summary, our results demonstrated that, following successful isolation of canine
9 D3 Q1 L1 A  D/ j+ f2 z2 ~transgenic cells, iSCNT embryos developed to early pre-implantation stages in vitro, showing stable7 @! k9 W# x) c0 J, o" S8 ~" y
GFP expression. These canine–bovine iSCNT embryos can be used for further in vitro analysis of canine2 m' h- o2 g3 h) ~( `0 V
transgenic cells and will contribute to the production of various transgenic dogs for use as specific
+ C  p( e  R9 u, r$ ohuman disease models.# q) E/ M8 J+ @8 j' Z* Z, F, ~$ F

4 }+ s2 f1 O) H9 y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。& i  M7 V2 N0 u$ v9 Q1 M# d; L