Generation of Cloned Transgenic Cats Expressing Red Fluorescence Protein1
Generation of Cloned Transgenic Cats Expressing Red Fluorescence Protein1
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DOI：
10.1095/biolreprod.107.065185
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发表时间：
2008-03
期刊：
影响因子：
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通讯作者：
Xi-jun Yin;Hyo-Sang Lee;Xian-Feng Yu;E. Choi;B. Koo;M. Kwon;Y. S. Lee;Su‐Jin Cho;G. Jin;L. Kim;H. Shin;Teoan Kim;Nam-Hyung Kim;I. Kong
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文献类型：
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作者：
Xi-jun Yin;Hyo-Sang Lee;Xian-Feng Yu;E. Choi;B. Koo;M. Kwon;Y. S. Lee;Su‐Jin Cho;G. Jin;L. Kim;H. Shin;Teoan Kim;Nam-Hyung Kim;I. Kong
摘要设计和生产转基因猫的方法对于建立人类疾病的生物医学模型是重要的。在这里，我们描述了利用体细胞核移植来产生系统表达红色荧光蛋白的克隆转基因猫。未成熟卵母细胞取自超数排卵的猫卵巢。供体成纤维细胞取自一只白色雄性土耳其安哥拉猫的耳皮肤活检组织，培养1~2代，并用设计用于转移和表达红色荧光蛋白(RFP)基因的逆转录病毒载体进行转导。将176枚RFP克隆胚胎移植给11个代孕母亲(平均每个受体16±7.5个)。3名代孕母亲成功受孕(27.3%)，并在怀孕65至66天时产下两只活产和一只死胎小猫。对9个猫科动物特有的微卫星基因座的分析证实，克隆猫与供体猫在基因上是相同的。通过聚合酶链式反应和Southern杂交分析，证实了RFP基因在转基因猫基因组中的存在。在活体出生的转基因猫(TG)出生60天后，检测到全身红色荧光，而在代孕母猫中没有检测到。在组织样本中检测到红色荧光，包括头发、肌肉、脑、心脏、肝脏、肾脏、脾、支气管、肺、胃、肠、舌头，甚至死胎TG猫的排泄物。这些结果表明，这种使用转基因体细胞的核移植程序可能有助于高效生产转基因猫。
Abstract A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases. Here we describe the use of somatic cell nuclear transfer to produce cloned transgenic cats that systemically express red fluorescent protein. Immature oocytes were collected from superovulating cat ovaries. Donor fibroblasts were obtained from an ear skin biopsy of a white male Turkish Angora cat, cultured for one to two passages, and subjected to transduction with a retrovirus vector designed to transfer and express the red fluorescent protein (RFP) gene. A total of 176 RFP cloned embryos were transferred into 11 surrogate mothers (mean = 16 ± 7.5 per recipient). Three surrogate mothers were successfully impregnated (27.3%) and delivered two liveborn and one stillborn kitten at 65 to 66 days of gestation. Analysis of nine feline-specific microsatellite loci confirmed that the cloned cats were genetically identical to the donor cat. Presence of the RFP gene in the transgenic cat genome was confirmed by PCR and Southern blot analyses. Whole-body red fluorescence was detected 60 days after birth in the liveborn transgenic (TG) cat but not in the surrogate mother cat. Red fluorescence was detected in tissue samples, including hair, muscle, brain, heart, liver, kidney, spleen, bronchus, lung, stomach, intestine, tongue, and even excrement of the stillborn TG cat. These results suggest that this nuclear transfer procedure using genetically modified somatic cells could be useful for the efficient production of transgenic cats.