Fu Hualin, Ishii Mamoru, Gu Ying, Maxson Robert
Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center and Hospital, University of Southern California Keck School of Medicine, Los Angeles, CA 90033, USA.
Genesis. 2007 Aug;45(8):477-81. doi: 10.1002/dvg.20316.
The msh-related homeobox genes, Msx1 and Msx2, have a variety functions during murine organogenesis, Msx1 in the development of the palate and teeth, Msx2 in the skull, teeth, and skin. Msx1 mutants die perinatally. Compound Msx1-2 mutants do not survive past late gestation. The multiplicity of functions of Msx1 and 2, as well as the lethality of Msx1 and Msx1-2 mutants limits the utility of the conventional knockouts. We therefore produced conditional alleles of Msx1 and Msx2. We constructed targeting vectors with LoxP sites flanking the homeodomain-encoding second exons and Frt sites flanking a neo gene. These vectors were used to produce targeted ES cells and mice with floxed alleles. The functionality of the LoxP sites in the floxed alleles was established by crosses with K14-Cre mice (epidermis-specific), and with an Msx2-Cre line that produces a germline deletion. Analysis of progeny by PCR revealed correct Cre-mediated recombination, as well as expected phenotypes.
与msh相关的同源框基因Msx1和Msx2在小鼠器官发生过程中具有多种功能,Msx1参与腭和牙齿的发育,Msx2参与颅骨、牙齿和皮肤的发育。Msx1突变体在出生时死亡。Msx1-2复合突变体在妊娠晚期后无法存活。Msx1和Msx2功能的多样性以及Msx1和Msx1-2突变体的致死性限制了传统基因敲除的实用性。因此,我们构建了Msx1和Msx2的条件性等位基因。我们构建了靶向载体,在编码同源结构域的第二个外显子两侧带有LoxP位点,在新霉素基因两侧带有Frt位点。这些载体用于产生带有floxed等位基因的靶向胚胎干细胞和小鼠。通过与K14-Cre小鼠(表皮特异性)以及产生种系缺失的Msx2-Cre品系杂交,确定了floxed等位基因中LoxP位点的功能。通过PCR对后代进行分析,揭示了正确的Cre介导的重组以及预期的表型。