Conditional knockout of Foxc2 gene in kidney: efficient generation of conditional alleles of single-exon gene by double-selection system
Conditional knockout of Foxc2 gene in kidney: efficient generation of conditional alleles of single-exon gene by double-selection system
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DOI：
10.1007/s00335-015-9610-y
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发表时间：
2016-02
期刊：
影响因子：
2.5
通讯作者：
M. Motojima;Sanae Ogiwara;T. Matsusaka;Sang Y. Kim;Nobuho Sagawa;K. Abe;M. Ohtsuka
中科院分区：
文献类型：
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作者：
M. Motojima;Sanae Ogiwara;T. Matsusaka;Sang Y. Kim;Nobuho Sagawa;K. Abe;M. Ohtsuka
Foxc2是一个单外显子基因，是包括肾脏在内的多个器官发育的关键调节因子。为了避免常规Foxc2敲除小鼠的胚胎致死性，我们在肾脏中有条件地删除Foxc2。单外显子基因的条件靶向涉及从启动子区域到编码区域的大的基因片段，以避免通过插入loxP位点对基因的功能破坏。因此，在传统的单一选择存活下来的胚胎干细胞克隆中，例如，只有新霉素耐药基因（neo），长柔段和目标基因组之间的同源重组导致只有一个loxP位点与选择标记相邻的发生率很高。为了避免这种限制，我们采用了双重选择系统。我们构建了Foxc2靶向构建体，其中一个固定片段包含4.6 kb的小鼠基因组和两个不同的选择标记基因，zeocin抗性基因和neo，它们被放置在每个loxP位点附近。经过zeocin和新霉素的双重选择，筛选出72个存活克隆，得到3个正确的靶向克隆。通过四倍体互补产生Foxc2小鼠后，我们将Dre和Flp重组酶的表达载体同时注射到体外受精卵中，从而去除两个选择标记基因。为了删除小鼠肾脏中的Foxc2，将Foxc2固定小鼠与Pax2-Cre小鼠交配。新生儿Pax2-Cre;Foxc2loxP/loxPmice表现为肾发育不全和肾小球囊肿。这些结果表明，采用双选择系统和单次微量注射同时去除选择标记制备Foxc2小鼠是可行的。
Foxc2 is a single-exon gene and a key regulator in development of multiple organs, including kidney. To avoid embryonic lethality of conventional Foxc2 knockout mice, we conditionally deleted Foxc2 in kidneys. Conditional targeting of a single-exon gene involves the large floxed gene segment spanning from promoter region to coding region to avoid functional disruption of the gene by the insertion of a loxP site. Therefore, in ES cell clones surviving a conventional single-selection, e.g., neomycin-resistant gene (neo) alone, homologous recombination between the long floxed segment and target genome results in a high incidence of having only one loxP site adjacent to the selection marker. To avoid this limitation, we employed a double-selection system. We generated a Foxc2 targeting construct in which a floxed segment contained 4.6 kb mouse genome and two different selection marker genes, zeocin-resistant gene andneo, that were placed adjacent to each loxP site. After double-selection by zeocin and neomycin, 72 surviving clones were screened that yielded three correctly targeted clones. After floxed Foxc2 mice were generated by tetraploid complementation, we removed the two selection marker genes by a simultaneous-single microinjection of expression vectors for Dre and Flp recombinases into in vitro-fertilized eggs. To delete Foxc2 in mouse kidneys, floxed Foxc2 mice were mated with Pax2-Cre mice. Newborn Pax2-Cre; Foxc2loxP/loxPmice showed kidney hypoplasia and glomerular cysts. These results indicate the feasibility of generating floxed Foxc2 mice by double-selection system and simultaneous removal of selection markers with a single microinjection.