Generation of brevican knockout mice / 第二军医大学学报

ABSTRACT

Objective To generate brevican knockout mutant mice. Methods The targeting construct to inactivate the brevican gene was made by using the ET cloning technology. The construct was linearized and electroporated into embryonic stem (ES) cells. Homologous recombinants were identifed by PCR after selecting the stable transfectants. The positive clones were then injected into C57BL/6 blastocysts, and the injected blastocysts were transferred into pseudopregnant foster mothers. Heterozygote and homozygote were multiplied by procreation mosaic. Progeny gene type was assayed by PCR. Results A neomycin resistance expression cassette under the control of the phosphoglycerate kinase (PGK) promoter was flanked by a 2.4-kb 5-arm fragment and a4.8-kb 3-arm fragment, thus introducing the NEO gene into exon 3 and deleting the encoding region from exon 3 to exon 8 after homologous recombination. Fourteen clones of gene-targeted ES cells were identified and three male chimeras with a higher than 50% chimeric ratio were produced. Six brevican-null mice were generated after outbred and inbred. Conclusion Genetically modified ES cells have been successfully generated by homologous recombination, and brevican-deficient mouse strain has been successfully generated.