Establishment and Identification of Liver-Specific CD36 Knockout Mice

doi:10.13523/j.cb.20180804

China Biotechnology

2018, Vol. 38

Issue (8): 26-33 DOI: 10.13523/j.cb.20180804

Establishment and Identification of Liver-Specific CD36 Knockout Mice

Chun-xiao SU,Xiao-yu ZHANG,Han ZENG,Ya-xi CHEN,Xiong-zhong RUAN,Ping YANG(

)

Centre for Lipid Research,Chongqing Key Laboratory of Lipid and Glucose Metabolism, Chongqing Medical University,Chongqing 400016,China

Download:

HTML

PDF(1112KB) HTML
Export: BibTeX | EndNote (RIS)

Abstract

Objective: To generate mice with a liver-specific knockout of CD36 gene using the Cre-Loxp system,which will lay a foundation for the study of the biological function of CD36. Methods: CD36 targeting vector was constructed and electroporated into embryonic stem cells.Positive clones with correct homologous recombination were screened by long-chain PCR.Positive embryonic stem cell clones were amplified and injected into the blastocysts of C57BL/6J mice to obtain chimeric mice.And then mated with Flp mice to obtain Flox heterozygous mice.The Flox mice were mated with the introduced Alb-Cre mice,and CD36 ^fl/fl:Alb-Cre ⁺ mice were obtained in the F3 generation,which are liver-specific CD36 knockout mice.The genotypes of the mice were identified by PCR.PCR,real-time fluorescence quantitative PCR and Western blot were used to verify the knockout effects of CD36 gene in the liver.The expression of CD36 in kidney,adipose tissue and myocardial tissue was detected by Western blot.Morphological changes of the liver were observed by HE staining. Results: Flox heterozygous mice with CD36 gene were established.After mating with Alb-Cre mice,CD36 ^fl/fl:Alb-Cre ^- and CD36 ^fl/fl:Alb-Cre ⁺ genotypes were screened in F3 generation.DNA levels confirmed that CD36 ^fl/fl:Alb-Cre ⁺ genotype mouse liver CD36 gene was knocked out by the Cre/Loxp recombinase system.Compared with mice with CD36 ^fl/fl:Alb-Cre ^- mice,CD36 ^fl/fl:Alb-Cre ⁺ mice had significantly reduced expression of CD36 mRNA and protein in the liver,and there was no difference in the expression of CD36 protein in kidney,adipose tissue and myocardial tissue.There was no significant difference in the morphological characteristics of liver. Conclusion: Liver-specific CD36 knockout mice were successfully generated by the Cre/Loxp recombinase system,providing an animal model for the study of CD36’s function in hepatic metabolism and diseases.

Key words： CD36 gene Gene knockout Cre/Loxp

Received: 17 April 2018 Published: 11 September 2018

ZTFLH:

Q819

Corresponding Authors: Ping YANG E-mail: 420577305@qq.com

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	Chun-xiao SU
	Xiao-yu ZHANG
	Han ZENG
	Ya-xi CHEN
	Xiong-zhong RUAN
	Ping YANG

Cite this article:

Chun-xiao SU,Xiao-yu ZHANG,Han ZENG,Ya-xi CHEN,Xiong-zhong RUAN,Ping YANG. Establishment and Identification of Liver-Specific CD36 Knockout Mice. China Biotechnology, 2018, 38(8): 26-33.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180804 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I8/26

Fig.1 Schematic diagram of CD36 gene targeting site and CD36 conditional gene targeting vector (a) The modes of CD36 targeting strategy (b) Diagram of the strategy to generate CD36 targeting vector

Fig.2 Strategy and procedure for construction of liver-specific CD36 knockout mouse model

Table 1 Genotyping Primer Information

Fig.3 Results of CD36 Conditional Gene Targeting Vector Identification, ES Cell Screening, and Flox Heterozygote Mouse Identification (a) The CD36 plasmid result of HindIII enzym (b) Results of 5' arm and 3' arm long range PCR (c) Identification of Flox heterozygous mice M:DNA marker V:CD36-cKO targeting vector E1,E2,E3,E4:homologous recombination positive ES cells C1,C2,C3,C4,C5:CD36 heterozygous mouse (CD36^fl/+)

Fig.4 Identification of mouse genotypes by agarose gel electrophoresis No. 1~4 mice were generated by mating CD36^fl/+ mice with Alb-Cre⁺ mice No. 5~8 mice were generated by mating CD36^fl/+:Alb-Cre⁺ mice with CD36^fl/fl mice; 1:CD36^+/+:Alb-Cre⁺; 2:CD36^+/+:Alb-Cre^-; 3:CD36^fl/+:Alb-Cre⁺; 4:CD36^fl/+:Alb-Cre^-; 5:CD36^fl/+:Alb-Cre^-; 6:CD36^fl/fl:Alb-Cre^-; 7:CD36^fl/+:Alb-Cre⁺; 8:CD36^fl/fl:Alb-Cre⁺

Fig.5 Genomic validation of liver specimens M:DNA marker

Fig.6 Liver CD36 mRNA expression in CD36^fl/fl: Alb-Cre^- and CD36^fl/fl:Alb-Cre⁺ mice (n=3) *:P<0.05

Fig.7 CD36 protein expression in CD36^fl/fl:Alb-Cre^- and CD36^fl/fl:Alb-Cre⁺ mice liver, kidney, adipose and myocardial tissue (a),(b),(c),(d) Expression of CD36 protein in liver, kidney, adipose and myocardial tissue were measured by Western blot (e)Protein band gray was scanned by Image J software according to Figure 7a,b,c,d *:P<0.05

Fig.8 Hepatic HE staining of CD36^fl/fl:Alb-Cre^-and CD36^fl/fl:Alb-Cre⁺ mice (10*20)


[1]	Gotoda T, Iizuka Y, Yamada N . Complex connection between CD36 and atherosclerosis,lipid metabolism,and insulin resistance syndromes. Curr Atheroscler Rep, 2000,2(6):453-454.

[2]	Pepino M Y, Kuda O, Samovski D , et al. Structure-function of CD36 and importance of fatty acid signal transduction in fat metabolism. Annu Rev Nutr, 2014,34(1):281-303. doi: 10.1146/annurev-nutr-071812-161220 pmid: 4329921

[3]	Canton J, Neculai D, Grinstein S . Scavenger receptors in homeostasis and immunity. Nat Rev Immunol, 2013,13(9):621-634. doi: 10.1038/nri3515 pmid: 23928573

[4]	Febbraio M, Hajjar D P, Silverstein R L . CD36: a class B scavenger receptor involved in angiogenesis,atherosclerosis,inflammation,and lipid metabolism. J Clin Invest, 2001,108(6):785-791. doi: 10.1172/JCI14006

[5]	Wilson C G, Tran J L, Erion D M , et al. Hepatocyte-specific disruption of CD36 attenuates fatty liver and improves insulin sensitivity in HFD fed mice. Endocrinology, 2016,157(2):570-585. doi: 10.1210/en.2015-1866

[6]	Pan X, Wang P, Luo J , et al. Adipogenic changes of hepatocytes in a high-fat diet-induced fatty liver mice model and non-alcoholic fatty liver disease patients. Endocrine, 2015,48(3):834-847. doi: 10.1007/s12020-014-0384-x

[7]	Nath A, Li I, Roberts L R , et al. Elevated free fatty acid uptake via CD36 promotes epithelial-mesenchymal transition in hepatocellular carcinoma. Sci Rep, 2015,5:14752. doi: 10.1038/srep14752

[8]	Liu P, Jenkins N A, Copeland N G . A highly efficient recombinerring-based method for generating conditional knockout mutations. Genome Res, 2003,13(3):476-484. doi: 10.1101/gr.749203

[9]	Chan W, Costantino N, Li R , et al. A recombineering based approach for high-throughput conditional knockout targeting vector construction. Nucleic Acids Res. 2007,35(8):e64. doi: 10.1093/nar/gkm163

[10]	孙一平, 王越, 金镇 , 等. SHBG基因敲除小鼠模型的建立及其表型分析.中国生物工程杂志. 2017,37(8):39-45. doi: 10.13523/j.cb.20170806

[10]	Sun Y P, Wang Y, Jin Z , et al. Establishment and phenotypic analysis of SHBG knockout mouse model. China Biotechnology, 2017,37(8):39-45. doi: 10.13523/j.cb.20170806

[11]	陈静静, 邢桂春, 张令强 . 基于Loxp/Cre系统的FBXL15基因敲除小鼠模型的建立.中国生物工程杂志. 2015,35(4):74-79. doi: 10.13523/j.cb.20150411

[11]	Chen J J, Xing G C, Zhang L Q . Establishment of FBXL15 knockout mouse model based on Loxp/Cre system. China Biotechnology, 2015,35(4):74-79. doi: 10.13523/j.cb.20150411

[12]	Koonen D P, Jacobs R L, Febbraio M , et al. Increased hepatic CD36 expression contributes to dyslipidemia associated with diet-induced obesity. Diabetes, 2007,56(12):2863-2871. doi: 10.2337/db07-0907

[13]	Luiken J J, Arumugam Y, Dyck D J , et al. Increased rates of fatty acid uptake and plasmalemmal fatty acid transporters in obese Zucker rats. J Biol Chem, 2001,276(44):40567-40573. doi: 10.1074/jbc.M100052200

[14]	Heeb?ll S, Poulsen M K, Ornstrup M J , et al. Circulating sCD36 levels in patients with non-alcoholic fatty liver disease and controls. Int J Obes, 2017,41(2):262-267. doi: 10.1038/ijo.2016.223 pmid: 27916988

[15]	Miquilena-Colina M E, Lima-Cabello E, Sánchez-Campos S , et al. Hepatic fatty acid translocase CD36 upregulation is associated with insulin resistance,hyperinsulinaemia and increased steatosis in non-alcoholic steatohepatitis and chronic hepatitis C. Gut, 2011,60(10):1394-1402. doi: 10.1136/gut.2010.222844

[16]	Currie E, Schulze A, Zechner R , et al. Cellular Fatty Acid Metabolism and Cancer. Cell Metabolism, 2013,18(2):153-161. doi: 10.1016/j.cmet.2013.05.017 pmid: 23791484

[17]	Pascual G, Avgustinova A, Mejetta S , et al. Targeting metastasis-initiating cells through the fatty acid receptor CD36. Nature, 2017,541(7635):41-45. doi: 10.1038/nature20791 pmid: 27974793

[18]	Stewart C R, Stuart L M, Wilkinson K , et al. CD36 ligands promote sterile inflammation through assembly of a Toll-like receptor 4 and 6 heterodimer. Nat Immunol, 2010,11(2):155-161. doi: 10.1038/ni.1836

[19]	Gao H, Volat F, Sandhow L , et al. CD36 Is a Marker of Human Adipocyte Progenitors with Pronounced Adipogenic and Triglyceride Accumulation Potential. Stem Cells, 2017,35(7), 1799-1814. doi: 10.1002/stem.v35.7

[20]	Schmidt-Supprian M, Rajewsky K . Vagaries of conditional gene targeting. Nat Immunol, 2007,8(7):665-668. doi: 10.1038/ni0707-665

[21]	Baker L G, Lodge J K . Multiple gene deletion in Cryptococcus neoformans using the Cre-lox system. Methods Mol Biol, 2012,845:85-98. doi: 10.1007/978-1-61779-539-8

[1]	GUO Sheng-nan, LI Xin-xiao, WANG Feng, LIU Kun-mei, DING Na, HU Qi-kuan, SUN Tao. *Establishment and Identification of the Neocortex and Hippocampus GABRG2* Knockout Mice and Its Preliminary Study in Generalized Epilepsy with Febrile Seizures Plus**[J]. China Biotechnology, 2020, 40(3): 9-20.

[2]	YU Chun-yang,ZHANG Chun,GUO Le,WAN Pan-pan,HUANG Yue,WANG Feng,LIU Kun-mei. *Construction of Hippocampal Cortical Specific Knockout AEG-1* Gene Mice and Preliminary Study on Its Behavior**[J]. China Biotechnology, 2020, 40(11): 10-20.

[3]	Chao-jing GUO,Qiong ZHU,Xin ZHANG,Lei LI,Ling-qiang ZHANG. *Generation and Phenotypic Analysis of Hepatic-specific Deubiquitinase OTUB1* Knockout Mice Model**[J]. China Biotechnology, 2019, 39(5): 80-87.

[4]	WAN Ying-han,CI Lei,WANG Jue,GONG Hui,LI Jun,DONG Ru,SUN Rui-lin,FEI Jian,SHEN Ru-ling. Construction and Preliminary Phenotypic Verification of PD-L1 Knockout Mice[J]. China Biotechnology, 2019, 39(12): 42-49.

[5]	WU Guo-guo,SONG Shu-ting,YUE Rong,ZHANG Jing,GUAN Ying,WANG Yue,LIU Bao-ai,LV Xue-min,WEI Jian-jun,ZHANG Hui-tu. Application of Counterseletable Gene upp in Genetic Manipulation of Streptomyces fungicidicus[J]. China Biotechnology, 2019, 39(11): 78-86.

[6]	LU Hai-yan,LI Jia-man,SUN Si-fan,ZHANG Xiao-mao,DING Juan-juan,ZOU Shao-lan. *Construction of an Auxotrophic Mutant from an Industrial Saccharomyces cerevisiae* Strain by CRISPR-Cas9 System**[J]. China Biotechnology, 2019, 39(10): 67-74.

[7]	Yu-rui SHENG,Bin LI,Bin WANG,Di ZUO,Lin MA,Xiao-fan REN,Le GUO,Kun-mei LIU. The Construction of AEG-1-Knockout U251 Cell Line by CRISPR/Cas9 Technology and Study of The Effect of AEG-1 on the Metastasis in U251 Cells[J]. China Biotechnology, 2018, 38(10): 38-47.

[8]	SUN Yi-ping, WANG Yue, JIN Zhen, WANG Xiao-yan, SUN Lei, ZHANG Xuan, FENG Chong, ZHOU Xiao-hua. *Establishment and Phenotype Analysis of SHBG* Knockout Mouse Model**[J]. China Biotechnology, 2017, 37(8): 39-45.

[9]	ZHANG Zhen-yang, YANG Yan-kun, ZHAN Chun-jun, LI Xiang, LIU Xiu-xia, BAI Zhong-hu. Pichia pastoris X-33 ΔGT2 Release the Glycerol Repression on AOX1 and Ef-ficiently Express Heterologous Proteins[J]. China Biotechnology, 2017, 37(1): 38-45.

[10]	DU Hong-yan, LI Tian-ming, LIU Jin-lei, FENG Hui-yong. *Construct the Uracil Phosphoribosyl Transferase Gene Mutant Strain in Gluconobacter suboxydans* for Seamless Genome Editing**[J]. China Biotechnology, 2016, 36(7): 64-71.

[11]	HAN Hai hong, WANG Jun qing, WANG Teng fei, XIAO Jing, HAN Deng lan, WANG Rui ming. *Method and Application of Gene Knockout Based Single Cross in Bacillus licheniformis* 20085**[J]. China Biotechnology, 2016, 36(11): 63-69.

[12]	CHANG Yu-mei, HOU Zhan-ming . Research on Gene Knockout and Function of FgPDE1 in Fusarium graminearum[J]. China Biotechnology, 2015, 35(8): 59-67.

[13]	SHEN Dong-ling, SHANG Shu-mei, LI Wei-na, YAN Jin-ping, HANGAN Ir-bis. *Characterization of the Disrupted ack* Genes on Fermentation by Thermoanaerobacterium calidifontis Rx1**[J]. China Biotechnology, 2015, 35(7): 37-44.

[14]	ZHANG Si-min, GAO Yue, FANG Yu-dan, ZHANG Jin-mai, ZHANG Jin-zhi. Construction of Mammary Gland-specific and Effective Expression Vector for Mammary Gland Bioreactor[J]. China Biotechnology, 2014, 34(7): 49-55.

[15]	TAO Si-mei, ZHENG Wei, ZHAO Peng-chao, ZHOU Wei, QUAN Chun-shan, FAN Sheng-di. *Effects of bmy* Gene knockout on Hemolysis and Antifungal activity of Bacillus amyloliquefaciens Q-426**[J]. China Biotechnology, 2014, 34(3): 56-60.

Viewed

Full text

Abstract

Cited

Shared

Discussed