Generation of Inducible HIF-1a RNAi Transgenic Mice

Abstract:

Hypoxia inducible factor-1α (HIF-1a) is a transcription factor that responds to changes in oxygen concentration. In this study, we constructed two vectors, HIF-AB and HIF-CD, to transcribe functional short interfering RNA against different region of mouse HIF-1 a. The oligonucleotide encoding small hairpin RNAs against mouse HIF-1 a was inserted into the downstream of U6 promoter of pBSK/U6-NEO plasmid. Cre-expression vector CRE-ERT2 with either HIF-AB or HIF-CD was transfected into RAW264.7 cell line, after selection with G418 and hygromycin, to obtain cell lines with stabilized expression of CRE-ERT2 and HIF-AB, or CRE-ERT2 and HIF-CD. The expression levels of HIF-1α of these stable cell lines were detected by semi-quantitative RT-PCR following treatment of CoCl2, a HIF-1 a expression inducer. The HIF-1 a mRNA expression was reduced by 85% and 72% by HIF-AB and HIF-CD respectively. HIF-AB vector was microinjected into pronucleus of zygotes to generate transgenic mice. We got two founder and two first filial generation transgenic mice containing HIF-AB and these transgenic mice were crossed with EIIA-Cre transgenic mouse to get EIIA-Cre;HIFRNAiflox/+ mice. The conditional knock down mice were viable and developed normally. The results of RT-PCR indicated that the HIF-1α mRNA expression of liver, lung and kidney were reduced significantly compared with the normal control.