Biggs Daniel, Chen Chiann-Mun, Davies Benjamin
Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK.
The Francis Crick Institute, London, UK.
Methods Mol Biol. 2023;2631:299-323. doi: 10.1007/978-1-0716-2990-1_13.
The targeting of transgenic constructs at single copy into neutral genomic loci avoids the unpredictable outcomes associated with conventional random integration approaches. The Gt(ROSA)26Sor locus on chromosome 6 has been used many times for the integration of transgenic constructs and is known to be permissive for transgene expression and disruption of the gene is not associated with a known phenotype. Furthermore, the transcript made from the Gt(ROSA)26Sor locus is ubiquitously expressed and subsequently the locus can be used to drive the ubiquitous expression of transgenes.Here we report a protocol for the generation of targeted transgenic alleles at Gt(ROSA)26Sor, taking as an example a conditional overexpression allele, by PhiC31 integrase/recombinase-mediated cassette exchange of an engineered Gt(ROSA)26Sor locus in mouse embryonic stem cells. The overexpression allele is initially silenced by the presence of a loxP flanked stop sequence but can be strongly activated through the action of Cre recombinase.
将转基因构建体以单拷贝形式靶向整合到中性基因组位点可避免与传统随机整合方法相关的不可预测结果。6号染色体上的Gt(ROSA)26Sor位点已被多次用于转基因构建体的整合，并且已知其允许转基因表达，该基因的破坏与已知表型无关。此外，由Gt(ROSA)26Sor位点产生的转录本在全身广泛表达，随后该位点可用于驱动转基因的全身表达。在此，我们报告了一种在Gt(ROSA)26Sor位点产生靶向转基因等位基因的方案，以条件性过表达等位基因为例，通过PhiC31整合酶/重组酶介导的盒式交换在小鼠胚胎干细胞中构建工程化的Gt(ROSA)26Sor位点。过表达等位基因最初因存在loxP侧翼的终止序列而沉默，但可通过Cre重组酶的作用被强烈激活。
