An efficient method for producing α(1,3)-galactosyltransferase gene knockout pigs
An efficient method for producing α(1,3)-galactosyltransferase gene knockout pigs
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DOI：
10.1089/clo.2004.6.327
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发表时间：
2004-12-01
期刊：
影响因子：
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通讯作者：
Nottle, M
中科院分区：
文献类型：
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作者：
Harrison, S;Boquest, A;Nottle, M
我们报道了一种相对有效的方法用于体细胞核移植和利用非等基因无启动子构建方法敲除猪胎儿成纤维细胞中的α(1，3)-半乳糖基转移酶(α1，3-GT)基因。在这里，我们报告了使用这些程序生产α1，3-GT基因敲除猪的情况。从108个新霉素抗性(neo(R))克隆中用长程PCR鉴定了7个α1，3-GT基因敲除细胞克隆，靶向效率为6.5%。用3个细胞克隆进行核移植。核移植采用先融合后激活的方案，使用的是体外成熟的成年卵母细胞。将51到110个融合的对联转移给10个受体，并在胚胎后1天同步进行。第115天引产，剖宫产仔猪。四名受试者共产下18头活猪。所有的猪都是雌性的，所有三个克隆都导致了活猪的出生。对α1，3-GT基因敲除猪进行长程聚合酶链式反应鉴定，并进行Southern杂交分析。我们的克隆方案的效率(胚胎移植/仔猪出生)对于所有移植的动物是1.9%，对于分娩的动物是4.6%。
We have reported relatively efficient methods for somatic cell nuclear transfer and for knocking out the alpha(1,3)-galactosyltransferase (alpha1,3-GT) gene in porcine fetal fibroblasts using a nonisogenic promoterless construct approach. Here we report the production of alpha1,3-GT gene knockout pigs using these procedures. Seven alpha1,3-GT gene knockout cell clones were identified by long-range PCR from 108 neomycin resistant (neo(R)) colonies, giving a 6.5% targeting efficiency. Three cell clones were used for nuclear transfer. Nuclear transfer was performed using a fusion before activation protocol using in vitro-matured adult oocytes. Between 51 and 110 fused couplets were transferred to 10 recipients synchronized 1 day behind the embryos. Parturition was induced on day 115, and piglets were delivered by caesarean section. Four recipients gave birth to a total of 18 live piglets. All pigs were female, and all three clones resulted in the birth of live pigs. alpha1,3-GT gene knockout pigs were identified by long-range PCR and confirmed by Southern blot analysis. The efficiency (embryos transferred/piglets born) of our cloning protocol was 1.9% for all transfers and 4.6% for animals that gave birth.