Jang Chuan-Wei, Behringer Richard R
Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA.
CSH Protoc. 2007 Oct 1;2007:pdb.prot4866. doi: 10.1101/pdb.prot4866.
INTRODUCTIONThis protocol describes a method for the production of transgenic rats by coinjecting circular PiggyBac transposon-containing plasmid DNA with transposase-encoding mRNA. After fertilized eggs are collected from females, pronuclei are microinjected with the DNA and mRNA and then transferred immediately into the oviducts of pseudopregnant foster females. Using this procedure, we have obtained transformation frequencies of 33%-100% (average of ~80%). Traditional methods for generating transgenic rats (i.e., microinjection of linear DNA) yield 3%-41% transgenic founders. Thus, transposon-mediated transformation of fertilized rat eggs is an efficient alternative method to generate transgenic rats.
引言
本方案描述了一种通过将含环形PiggyBac转座子的质粒DNA与编码转座酶的mRNA共注射来制备转基因大鼠的方法。从雌性大鼠收集受精卵后，将DNA和mRNA显微注射到原核中，然后立即将其转移到假孕代孕雌性大鼠的输卵管中。使用该程序，我们获得了33%-100%（平均约80%）的转化频率。传统的制备转基因大鼠的方法（即线性DNA显微注射）产生3%-41%的转基因奠基者。因此，转座子介导的大鼠受精卵转化是一种高效的制备转基因大鼠的替代方法。
