Germ-line transmission of lentiviral PGK-EGFP integrants in transgenic cattle: new perspectives for experimental embryology
Germ-line transmission of lentiviral PGK-EGFP integrants in transgenic cattle: new perspectives for experimental embryology
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DOI：
10.1007/s11248-009-9333-5
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发表时间：
2010-08-01
影响因子：
3
通讯作者：
Wolf, Eckhard
中科院分区：
文献类型：
--
作者：
Reichenbach, Myriam;Lim, Tiongti;Wolf, Eckhard
慢病毒载体是家畜基因改造的有力工具。我们以前用慢病毒整合体在磷酸甘油酸激酶(PGK)启动子的控制下获得了携带增强型绿色荧光蛋白(EGFP)的转基因方正牛。在这项研究中，我们研究了LV-PGK-EGFP整合子在牛的雌性和雄性生殖系中的传播。一头转基因小母牛(#562，Kiki)接受了超数排卵治疗，并用非转基因公牛的精液授精。胚胎被回收并转移到同步的受体小母牛，通过体内成像检测，导致了一头健康的表达EGFP的雄性转基因小牛的出生。用转基因方正公牛(#561，Jojo)的精液对非转基因母牛的体外成熟卵母细胞进行体外受精。体外卵裂率和囊胚率分别为52.0+/-A4.1和24.5+/-A4.4%。在囊胚期(体外受精后第7天)可观察到EGFP的表达，93.0%(281/302)的胚胎有EGFP的表达。将24个表达EGFP的胚胎移植给9个同步受体。对15天从子宫中冲出的2个胚胎、45天恢复的2个胎儿和1头健康的雄性转基因牛犊的分析表明，所有被调查的组织中都有持续的高水平表达EGFP。我们的研究首次表明，慢病毒整合体通过雌性和雄性转基因方正牛的胚系传播。继承模式符合孟德尔规则。重要的是，方正#561在胚胎、胎儿和后代中的高保真表达为牛的发育研究提供了有趣的工具，包括配子、胚胎和胎儿与其母体环境的相互作用。
Lentiviral vectors are a powerful tool for the genetic modification of livestock species. We previously generated transgenic founder cattle with lentiviral integrants carrying enhanced green fluorescent protein (EGFP) under the control of the phosphoglycerate kinase (PGK) promoter. In this study, we investigated the transmission of LV-PGK-EGFP integrants through the female and male germ line in cattle. A transgenic founder heifer (#562, Kiki) was subjected to superovulation treatment and inseminated with semen from a non-transgenic bull. Embryos were recovered and transferred to synchronized recipient heifers, resulting in the birth of a healthy male transgenic calf expressing EGFP as detected by in vivo imaging. Semen from a transgenic founder bull (#561, Jojo) was used for in vitro fertilization (IVF) of in vitro matured (IVM) oocytes from non-transgenic cows. The rates of cleavage and development to blastocyst in vitro corresponded to 52.0 +/- A 4.1 and 24.5 +/- A 4.4%, respectively. Expression of EGFP was observed at blastocyst stage (day 7 after IVF) and was seen in 93.0% (281/302) of the embryos. 24 EGFP-expressing embryos were transferred to 9 synchronized recipients. Analysis of 2 embryos, flushed from the uterus on day 15, two fetuses recovered on day 45, and a healthy male transgenic calf revealed consistent high-level expression of EGFP in all tissues investigated. Our study shows for the first time transmission of lentiviral integrants through the germ line of female and male transgenic founder cattle. The pattern of inheritance was consistent with Mendelian rules. Importantly, high fidelity expression of EGFP in embryos, fetuses, and offspring of founder #561 provides interesting tools for developmental studies in cattle, including interactions of gametes, embryos and fetuses with their maternal environment.