Establishment of a mouse model with liver-specific HIF-2α gene knockout with Cre-loxP technique

doi:10.3877/cma.j.issn.2095-3232.2019.03.021

Abstract

Abstract:

Objective

To investigate the feasibility of establishing a mouse model with liver-specific HIF-2α gene knockout using Cre-LoxP technique.

Methods

The loxP-labeled mice with HIF-2α gene were introduced from the Jackson Laboratory of the United States. A liver-specific HIF-2α gene knockout mouse model was established by using Cre/loxP recombinase system and Alb-Cre transgenic mice through multiple generations of hybridization. The genotypes of mouse were identified by PCR. The expression levels of HIF-2α mRNA in the liver, fat and muscular tissues were detected by RT-qPCR. The blood glucose, blood lipid and liver function levels in the wild-type mice and Alb-Cre⁺/HIF-2α^fl/fl homozygous mice were detected. The levels of HIF-2α mRNA among three groups were statistically compared by one-way ANOVA and Turkey test.

Results

The genotypes of the 2^nd generation mouse were successfully identified, including Alb-Cre⁺/HIF-2α^fl/fl and Alb-Cre^-/HIF-2α^fl/fl homozygous mice. The expression level of HIF-2α mRNA in the liver tissues of HIF-2α gene knockout mice was 0.10±0.02, significantly lower than 1.00±0.10 of the wild-type mice (HSD=-1.87, P<0.05).

Conclusion

The mouse models with liver-specific HIF-2α gene knockout can be successfully established using Cre-LoxP technique.

Key words: Hypoxia-inducible factor 2, alpha subunit, Mice, knockout, Cre/loxP recombinase system, Fatty liver, nonalcoholic

Anzhu Zhao, Huirong Fu, Yun Li, Jiandi Chen, Hongyun Lu. Establishment of a mouse model with liver-specific HIF-2α gene knockout with Cre-loxP technique[J]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2019, 08(03): 270-275.

/ / Recommend

Add to citation manager EndNote|Ris|BibTeX

URL: https://zhgzwkssxdzzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.2095-3232.2019.03.021

https://zhgzwkssxdzzz.cma-cmc.com.cn/EN/Y2019/V08/I03/270

Figures/Tables 7

References 18

[1]	Aron-Wisnewsky J,Minville C,Tordjman J, et al. Chronic intermittent hypoxia is a major trigger for non-alcoholic fatty liver disease in morbid obese[J]. Hepatol, 2012, 56(1):225-233.
[2]	Nishiyama Y,Goda N,Kanai M, et al. HIF-1α induction suppresses excessive lipid accumulation in alcoholic fatty liver in mice[J]. Hepatol, 2012, 56(2):441-447.
[3]	Loboda A,Jozkowicz A,Dulak J. HIF-1 and HIF-2 transcription factors--similar but not identical[J]. Mol Cells, 2010, 29(5):435-442.
[4]	De Minicis S,Day C,Svegliati-Baroni G. From NAFLD to NASH and HCC: pathogenetic mechanisms and therapeutic insights[J]. Curr Pharmaceut Des, 2013, 19(29):5239-5249.
[5]	Rosmorduc O,Fartoux L. HCC and NASH: how strong is the clinical demonstration[J]. Clin Res Hepatol Gastroenterol, 2012, 36(3):202-208.
[6]	Said A,Ghufran A. Epidemic of non-alcoholic fatty liver disease and hepatocellular carcinoma[J]. World J Clin Oncol, 2017, 8(6):429-436.
[7]	Alexander J,Torbenson M,Wu TT, et al. Non-alcoholic fatty liver disease contributes to hepatocarcinogenesis in non-alcoholic liver: a clinical and pathological study[J]. J Gastroenterol Hepatol, 2013, 28(5):848-854.
[8]	Sanyal A,Poklepovic A,Moyneur E, et al. Population-based risk factors and resource utilization for HCC: US perspective[J]. Curr Med Res Opin, 2010, 26(9):2183-2191.
[9]	Younossi ZM,Otgonsuren M,Henry L, et al．Association of nonalcoholic fatty liver disease (NAFLD) with hepatocellular carcinoma (HCC) in the United States from 2004 to 2009[J]. Hepatology, 2015, 62(6):1723-1730．
[10]	Singal AG,El-Serag HB．Hepatocellular carcinoma from epidemiology to prevention: translating knowledge into practice[J]．Clin Gastroenterol Hepatol, 2015, 13(12):2140-2151.
[11]	Younossi ZM,Koenig AB,Abdelatif D, et al. Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes[J]. Hepatology, 2016, 64(1):73-84.
[12]	Estes C,Razavi H,Loomba R, et al. Modeling the epidemic of nonalcoholic fatty liver disease demonstrates an exponential increase in burden of disease[J]. Hepatology, 2018, 67(1):123-133.
[13]	Turnbull CD. Intermittent hypoxia, cardiovascular disease and obstructive sleep apnoea[J]. J Thorac Dis, 2018, 10(Suppl 1):S33-39.
[14]	Liu Y,Ma Z,Zhao C, et al. HIF-1α and HIF-2α are critically involved in hypoxia-induced lipid accumulation in hepatocytes through reducing PGC-1α-mediated fatty acid β-oxidation[J]. Toxicol Lett, 2014, 226(2):117-123.
[15]	Cao R,Zhao X,Li S, et al. Hypoxia induces dysregulation of lipid metabolism in HepG2 cells via activation of HIF-2α[J]. Cell Physiol Biochem, 2014, 34(5):1427-1441.
[16]	Baguet JP,Barone-Rochette G,Tamisier R, et al. Mechanisms of cardiac dysfunction in obstructive sleep apnea[J]. Nat Rev Cardiol, 2012, 9(12):679-688.
[17]	Yang SL,Wu C,Xiong ZF, et al. Progress on hypoxia-inducible factor-3: its structure, gene regulation and biological function (review)[J]. Mol Med Rep, 2015, 12(2):2411-2416.
[18]	Ramakrishnan SK,Shah YM. A central role for hypoxia-inducible factor (HIF)-2α in hepatic glucose homeostasis[J]. Nutr Healthy Aging, 2017, 4(3): 207-216.

名称	引物	引物类型	引物序列	产物（bp）
Alb-Cre	Primer Ⅰ	突变型上游引物	ATTTGCCTGCATTACCGGTCG	300～400
?	Primer Ⅱ	突变型下游引物	CAGCATTGCTGTCACTTGGTC	?
?	Control-P1	突变型上游引物	CAAATGTTGCTTGTCTGGTG	200
?	Control-P2	突变型下游引物	GTCAGTCGAGTGCACAGTTT	?
HIF-2α	olMR8468	上游引物	GAGAGCAGCTTCTCCTGGAA	MT:220 WT:182
?	olMR8469	下游引物	TGTAGGCAAGGAAACCAAGG	?

名称	引物	引物类型	引物序列	产物（bp）
Alb-Cre	Primer Ⅰ	突变型上游引物	ATTTGCCTGCATTACCGGTCG	300～400
?	Primer Ⅱ	突变型下游引物	CAGCATTGCTGTCACTTGGTC	?
?	Control-P1	突变型上游引物	CAAATGTTGCTTGTCTGGTG	200
?	Control-P2	突变型下游引物	GTCAGTCGAGTGCACAGTTT	?
HIF-2α	olMR8468	上游引物	GAGAGCAGCTTCTCCTGGAA	MT:220 WT:182
?	olMR8469	下游引物	TGTAGGCAAGGAAACCAAGG	?

基因名称	引物	引物类型	引物序列	产物（bp）
HIF-2α	P1	上游引物	CTCCCACCTGGACAAAGC	18
?	P2	下游引物	GTCGCAAGGATGAGTGAAGT	20
Actin	P3	上游引物	TTGCTGACAGGATGCAGAAGGAGA	24
?	P4	下游引物	ACTCCTGCTTGCTGATCCACATCT	24

基因名称	引物	引物类型	引物序列	产物（bp）
HIF-2α	P1	上游引物	CTCCCACCTGGACAAAGC	18
?	P2	下游引物	GTCGCAAGGATGAGTGAAGT	20
Actin	P3	上游引物	TTGCTGACAGGATGCAGAAGGAGA	24
?	P4	下游引物	ACTCCTGCTTGCTGATCCACATCT	24

Please choose a citation manager

Content to export