[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& w# s' A- |2 ^! u
cell nuclear transfer
- S9 w4 N' b# I) s3 x6 NSo Gun Hong2, Hyun Ju Oh2, Jung Eun Park2, Min Jung Kim2, Geon A. Kim2, Ok Jae Koo2,7 C# v8 }2 b7 a5 j: @6 z
Goo Jang2 and Byeong Chun Lee17 R: R; m4 V3 a" f( X7 i0 f$ J
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Gwanak-gu,8 X5 I- q: Y4 I2 F& Z. |
Seoul, Korea3 i& k/ e% F4 N8 ?3 \
Date submitted: 10.03.2010. Date accepted: 29.09.2010
5 Z$ W. n, d/ q! ZSummary, h4 K& v: O! g" C- I+ A
Somatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals
' {9 ?& T0 E( yand deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured0 H! k4 ]0 L, H$ [
oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells. B5 B; S3 m! p; J
carry ‘foreign’ DNA and are clones when all the donor cells are genetically identical. However, in1 J$ y# y9 Z( I- z6 l1 x1 O
canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency
- {7 K4 m5 @$ x( h) e2 Z% ?+ M7 X2 Iof in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is" y2 O8 P4 u  Y) ^
to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be
$ B! _" |& m/ B2 q$ pmatured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast2 v, U4 E* z  Y; G8 Y/ K5 B- j
line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic
& G% w/ r% E, [development of canine cloned embryos derived from transgenic and non-transgenic cell lines using2 m# {: B2 L1 z9 P
bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the
. i* E5 w% ]: N6 v3 m+ xGFP and puromycin resistance genes using FuGENE 6 R  . Viability levels of these cells were determined5 t* K+ O9 p- S( Q4 D0 g4 O5 {; ~
by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT' S* U$ u. J. e
(iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT5 O; b. k2 S' f. z/ A# u- x
measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different
; g: P, ]; d5 p! |/ X8 pfrom non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic9 c1 O2 Q0 e( ?. q' m
iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively),
$ I; P3 c  t0 M, U, G9 j8 _; o+ v9 icleavage rates (69.7% vs. 73.8%) and development to the 8–16-cell stage (40.1% vs. 42.7%). Embryos! N, K9 L) r8 Y8 @
derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8–16-cell stages' t! S, Y% B. N# W) Z$ N; {' A8 }9 ~
without mosaicism. In summary, our results demonstrated that, following successful isolation of canine
4 m) {0 C3 l5 p$ v- M( Mtransgenic cells, iSCNT embryos developed to early pre-implantation stages in vitro, showing stable! V$ M  i! u. J1 L; w% C; L" v
GFP expression. These canine–bovine iSCNT embryos can be used for further in vitro analysis of canine
8 s  K" E' |" M) Rtransgenic cells and will contribute to the production of various transgenic dogs for use as specific* W  K( y0 \0 T2 A
human disease models.
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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。
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