Sendai Yutaka, Sawada Tokihiko, Urakawa Manami, Shinkai Yoichi, Kubota Keiichi, Hoshi Hiroyoshi, Aoyagi Yoshito
Research Institute for the Functional Peptides, Yamagata, Japan.
Transplantation. 2006 Mar 15;81(5):760-6. doi: 10.1097/01.tp.0000190422.66657.f1.
Gene targeting in large animals has the potential to be useful in medicine as well as in agriculture. Previously, we reported the first successful targeting of the bovine alpha1,3-galactosyltransferase (alpha1,3GT) gene and establishment of a heterozygous knockout cell line. In this report, we generated both heterozygous and homozygous knockout bovine cell lines, and alpha1,3GT-gene knockout cattle.
alpha1,3GT gene-disruption was accomplished using primary fetal fibroblasts with a single targeting vector, a promoter-less positive selection vector containing IRES (internal ribosome entry site)-antibiotic-resistance gene (neo) cassette and loxP sequences. At each step in establishing heterozygous and homozygous knockout cell lines, the antibiotic-resistance gene cassette in the targeted allele was removed by a Cre-loxP recombination system that utilizes an adenovirus with transient Cre recombinase expression. A nuclear transfer was performed using alpha1,3GT fetal fibroblasts, and one alpha1,3GT knockout calf was generated but died shortly after birth (day 287).
Necropsy revealed normal morphology in all organs. The calf weighed 22.3 kg at birth and this value is within the normal range.
The alpha1,3GT knockout- and antibiotic-resistance gene free (alpha1,3GT(-/-)neo-) cells could be cloned normally. Thus, cloned cattle from alpha1,3GT(-/-) neo- cells are potentially safer for human use. Additionally, our strategy is faster and more economical than backcrossing to produce homozygous knockouts. This method should be useful for future production of knockouts of multiple genes in livestock.
在大型动物中进行基因靶向操作在医学和农业领域都具有潜在的应用价值。此前，我们报道了首次成功靶向牛α1,3-半乳糖基转移酶（α1,3GT）基因并建立了杂合敲除细胞系。在本报告中，我们生成了杂合和纯合敲除牛细胞系以及α1,3GT基因敲除牛。
使用带有单个靶向载体的原代胎儿成纤维细胞实现α1,3GT基因破坏，该靶向载体是一个无启动子的阳性选择载体，包含内部核糖体进入位点（IRES）-抗生素抗性基因（neo）盒和loxP序列。在建立杂合和纯合敲除细胞系的每个步骤中，利用表达瞬时Cre重组酶的腺病毒通过Cre-loxP重组系统去除靶向等位基因中的抗生素抗性基因盒。使用α1,3GT胎儿成纤维细胞进行核移植，生成了一头α1,3GT敲除小牛，但出生后不久（第287天）死亡。
尸检显示所有器官形态正常。这头小牛出生时体重为22.3千克，该数值在正常范围内。
α1,3GT敲除且无抗生素抗性基因（α1,3GT(-/-)neo-）的细胞能够正常克隆。因此，来自α1,3GT(-/-) neo-细胞的克隆牛对人类使用可能更安全。此外，我们的策略比回交产生纯合敲除更快且更经济。该方法对于未来家畜中多个基因敲除的生产应该是有用的。
