Dang Su-Ying, Ma Sun-Kai, Sun Xia, Yan Lan-Zhen, Wang Zhu-Gang
Shanghai Research Center for Model Organisms, Shanghai 201203, China.
Sheng Wu Gong Cheng Xue Bao. 2005 Jan;21(1):159-62.
To generate transgenic mice in which both hygromycin (hyg) and neomycin (neo) resistance genes are expressed in murine fibroblast cells (MEFs), which are required for conditional gene knock-out and screening of drug resistant ES cell clones. To construct HygR-neoR expression vector, pTK-hygR-pA and PGK-neoR-pA were cloned into pBluescript vector. DNA fragments of tandem genes ( 4245bp ) were prepared by Kpn I and Xba I digestion and transgene was microinjected into pronucleus of zygotes to generate transgenic mice. Transgenic mice were identified by PCR and Southern blot; expression of hygR and neoR gene transcripts were detected by RT-PCR. 7 founder mice carrying hyg-neo resistant genes were obtained and 6 transgenic mouse lines were successfully established. The hygR and neoR gene transcripts were detected in the liver and/or ovary of transgenic mice from hn30, hn33, hn66 and hn67 mouse lines. In MEFs isolated from the mice of line hn66 and hn30, expression of hyg and neo resistant genes was also detectable. Transgenic mouse lines expressing two anti-drug genes have been established. The hyg and neo resistant gene transcripts were detected in the MEFs of two transgenic mouse lines.
为了培育出在鼠成纤维细胞（MEFs）中表达潮霉素（hyg）和新霉素（neo）抗性基因的转基因小鼠，这些细胞是条件性基因敲除和耐药性胚胎干细胞（ES）克隆筛选所必需的。为构建HygR-neoR表达载体，将pTK-hygR-pA和PGK-neoR-pA克隆到pBluescript载体中。通过Kpn I和Xba I酶切制备串联基因的DNA片段（4245bp），并将转基因显微注射到受精卵的原核中以产生转基因小鼠。通过PCR和Southern印迹鉴定转基因小鼠；通过RT-PCR检测hygR和neoR基因转录本的表达。获得了7只携带hyg-neo抗性基因的奠基小鼠，并成功建立了6个转基因小鼠品系。在来自hn30、hn33、hn66和hn67小鼠品系的转基因小鼠的肝脏和/或卵巢中检测到hygR和neoR基因转录本。在从hn66和hn30品系小鼠分离得到的MEFs中，也可检测到hyg和neo抗性基因的表达。已建立了表达两种抗药基因的转基因小鼠品系。在两个转基因小鼠品系的MEFs中检测到了hyg和neo抗性基因转录本。
