Production of transgenic calves expressing an shRNA targeting myostatin.
Production of transgenic calves expressing an shRNA targeting myostatin.
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DOI：
10.1002/mrd.22007
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发表时间：
2012-03
影响因子：
2.5
通讯作者：
Westhusin, M. E.
中科院分区：
文献类型：
--
作者：
Tessanne, K.;Golding, M. C.;Long, C. R.;Peoples, M. D.;Hannon, G.;Westhusin, M. E.
肌肉生长抑制素(MSTN)是一种众所周知的肌肉生长负调控因子。拥有该基因突变的动物表现出增强的肌肉表型，这是一种理想的农业特征。然而，新生儿发病率增加是常见的，这是由于胎儿肌肉质量增加而导致的后代出生时出现的并发症。目前这项研究的目的是在大型动物模型中使用RNA干扰来产生减弱的MSTN缺失表型，以促进肌肉发育，而不会产生失活突变的有害后果。为此，我们鉴定了一系列短干扰RNA，表明它们有效地抑制了MSTN的mRNA和蛋白水平。为了在体内获得稳定抑制MSTN的转基因后代，将针对MSTN的短发夹状RNA(ShRNA)的重组慢病毒载体导入牛胎儿成纤维细胞。这些细胞被用作体细胞核移植(SCNT)的核供体。将20个囊胚移植到7头受体奶牛体内，结果有5头怀孕。一头转基因小牛发育到足月，但在剖腹产后死亡。作为一种替代策略，将重组慢病毒颗粒显微注射到体外产生的牛受精卵的卵黄周间隙中，产生了40个转基因囊胚，这些囊胚被转移到14头受体牛身上，导致7次怀孕。共产生了5头转基因小牛，其中3头表达了转基因。这是通过直接注射重组慢病毒并表达针对内源基因(Myostatin)的shRNAs的转基因家畜的第一份报告。
Myostatin (MSTN) is a well-known negative regulator of muscle growth. Animals that possess mutations within this gene display an enhanced muscling phenotype, a desirable agricultural trait. Increased neonatal morbidity is common, however, resulting from complications arising from the birth of offspring with increased fetal muscle mass. The objective of the current research was to generate an attenuated MSTN-null phenotype in a large-animal model using RNA interference to enhance muscle development without the detrimental consequences of an inactivating mutation. To this end, we identified a series of short interfering RNAs that demonstrated effective suppression of MSTN mRNA and protein levels. To produce transgenic offspring capable of stable MSTN suppression in vivo, a recombinant lentiviral vector expressing a short hairpin RNA (shRNA) targeting MSTN for silencing was introduced into bovine fetal fibroblasts. These cells were used as nucleus donors for somatic cell nuclear transfer (SCNT). Twenty blastocysts were transferred into seven recipient cows resulting in five pregnancies. One transgenic calf developed to term, but died following delivery by Caesarean-section. As an alternative strategy, microinjection of recombinant lentiviral particles into the perivitelline space of in vitro-produced bovine zygotes was utilized to produce 40 transgenic blastocysts that were transferred into 14 recipient cows, resulting in 7 pregnancies. Five transgenic calves were produced, of which three expressed the transgene. This is the first report of transgenic livestock produced by direct injection of a recombinant lentivirus, and expressing transgenes encoding shRNAs targeting an endogenous gene (myostatin) for silencing.
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影响因子：
2.1
作者：
Cassar-Malek, I;Langlois, N;Geay, Y
通讯作者：
Geay, Y
影响因子：
4.5
作者：
Mosher DS;Quignon P;Bustamante CD;Sutter NB;Mellersh CS;Parker HG;Ostrander EA
通讯作者：
Ostrander EA
影响因子：
3
作者：
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通讯作者：
Boesze, Zsuzsanna
影响因子：
1.7
作者：
Milazzotto, Marcella Pecora;Goissis, Marcelo Demarchi;Visintin, Jose Antonio
通讯作者：
Visintin, Jose Antonio
影响因子：
7.7
作者：
Hofmann, A;Kessler, B;Pfeifer, A
通讯作者：
Pfeifer, A