Insertional Mutagenesis by a Hybrid PiggyBac and Sleeping Beauty Transposon in the Rat
Insertional Mutagenesis by a Hybrid PiggyBac and Sleeping Beauty Transposon in the Rat
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DOI：
10.1534/genetics.112.140855
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发表时间：
2012-12-01
期刊：
影响因子：
3.3
通讯作者：
Behringer, Richard R.
中科院分区：
文献类型：
--
作者：
Furushima, Kenryo;Jang, Chuan-Wei;Behringer, Richard R.
在大鼠中建立了一个基于piggyBac/Sleeping Beauty转座子的插入突变系统，该系统可以通过简单的育种来动员。这些转座子被改造为包括基因陷阱序列和酪氨酸酶（Tyr）色素沉着报告基因，以拯救诱变策略中使用的遗传背景的白化病。通过将携带转座子的质粒和编码piggyBac转座酶的RNA共注射到合子中，将单拷贝转座子插入转座到大鼠基因组中。转基因Tyr表达水平受染色体环境影响，导致转基因大鼠具有不同的色素沉着，从而能够进行视觉基因分型。通过标准受精卵注射也在白化病背景下产生设计为普遍表达piggyBac或睡美人转座酶的转基因大鼠。在已知位点携带单拷贝转座子和转座酶转基因的双基因大鼠表现出毛色镶嵌，表明体细胞转座。PiggyBac或睡美人转座酶双基因大鼠与野生型白化病大鼠交配产生的后代，其色素沉着与最初的转座子插入不同，这是种系转座到新基因座的结果。睡美人和piggyBac的生殖系转座频率类似于10%或每窝约一个新插入。大约50%的插入发生在内含子中。嵌合体转录本含有内源性和基因陷阱序列的Gabrb 1突变大鼠。这种基于简单杂交和视觉基因分型的诱变系统可用于在大鼠中产生单基因突变的集合。
A hybrid piggyBac/Sleeping Beauty transposon-based insertional mutagenesis system that can be mobilized by simple breeding was established in the rat. These transposons were engineered to include gene trap sequences and a tyrosinase (Tyr) pigmentation reporter to rescue the albinism of the genetic background used in the mutagenesis strategy. Single-copy transposon insertions were transposed into the rat genome by co-injection of plasmids carrying the transposon and RNA encoding piggyBac transposase into zygotes. The levels of transgenic Tyr expression were influenced by chromosomal context, leading to transgenic rats with different pigmentation that enabled visual genotyping. Transgenic rats designed to ubiquitously express either piggyBac or Sleeping Beauty transposase were generated by standard zygote injection also on an albino background. Bigenic rats carrying single-copy transposons at known loci and transposase transgenes exhibited coat color mosaicism, indicating somatic transposition. PiggyBac or Sleeping Beauty transposase bigenic rats bred with wild-type albino rats yielded offspring with pigmentation distinct from the initial transposon insertions as a consequence of germline transposition to new loci. The germline transposition frequency for Sleeping Beauty and piggyBac was similar to 10% or about one new insertion per litter. Approximately 50% of the insertions occurred in introns. Chimeric transcripts containing endogenous and gene trap sequences were identified in Gabrb1 mutant rats. This mutagenesis system based on simple crosses and visual genotyping can be used to generate a collection of single-gene mutations in the rat.