利用CRISPR/Cas9 AAV系统构建纹状体Slc20a2基因敲除小鼠模型

doi:10.16288/j.yczz.20-138

[an error occurred while processing this directive]

Hereditas(Beijing) ›› 2020, Vol. 42 ›› Issue (10): 1017-1027.doi: 10.16288/j.yczz.20-138

• Research Article • Previous Articles Next Articles

Construction of a striatum-specific Slc20a2 gene knockout mice model by CRISPR/Cas9 AAV system

Minting Lin^1,², Lulu Lai¹, Miao Zhao¹, Biwei Lin¹, Xiangping Yao^1,³()

^1. Department of Neurology, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, China
^2. Institute of Neurosciences, Fujian Medical University, Fuzhou 350004, China
^3. Fujian Key Laboratory of Molecular Neurology, Fuzhou 350005, China

Received:2020-05-18 Revised:2020-08-18 Online:2020-10-20 Published:2020-10-12
Contact: Yao Xiangping E-mail:119373522@qq.com
Supported by:
Supported by the National Natural Science Foundation of China No)(81801129);Startup Fund for Scientific Research of Fujian Medical University Nos(2017XQ1071);Startup Fund for Scientific Research of Fujian Medical University Nos(2018QH2035)

Abstract

Abstract:

Primary familial brain calcification (PFBC) is a chronic progressive neurogenetic disorder. Its clinical symptoms mainly include dyskinesia, cognitive disorder and mental impairment; and the pathogenesis remains unclear. Studies have shown that SLC20A2 is the most common pathogenic gene of the disease. Since the Slc20a2 gene knockout mouse model could result in fetal growth restriction, in order to better understand the pathogenesis of PFBC, the present study used the CRISPR/Cas9 technology to construct a conditional knockout model of Slc20a2 gene in the striatum of mice. First, three sgRNAs (single guide RNAs) were designed to target the exon3 of Slc20a2 gene. The activity of the respective sgRNA was verified by constructing expression plasmids, transfecting cells and Surveyor assay. Second, the SgRNA with the highest activity was selected to generate the recombinant AAV-Cre virus, which was injected into the striatum of mice by stereotactic method. In vitro experiments showed that the three sgRNAs could effectively mediate Cas9 cleavage of the respective target DNA. The activity of Cre recombinase of the AAV-Cre was confirmed by immunofluorescence assay. Immunohistochemistry, TA clone, high-throughput sequencing and Western blot were used to detect and evaluate the efficiency of Slc20a2 gene knockout. The results showed that the Slc20a2 expression in the striatum of mice in the experimental group decreased significantly. In this study, three sgRNAs capable of knockout of Slc20a2 were successfully designed, and the conditional knockout of the Slc20a2 gene in the striatum of mouse was successfully established by the CRISPR/Cas9 technology, thereby providing an effective animal model for studying the pathogenesis of PFBC.

Key words: CRISPR/Cas9, Slc20a2, sgRNA, gene knockout, mice model

Minting Lin, Lulu Lai, Miao Zhao, Biwei Lin, Xiangping Yao. Construction of a striatum-specific Slc20a2 gene knockout mice model by CRISPR/Cas9 AAV system[J]. Hereditas(Beijing), 2020, 42(10): 1017-1027.

Figures/Tables 6

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

References 21

[1]	Nicolas G, Pottier C, Charbonnier C, Guyant-Marechal L, Le Ber I, Pariente J, Labauge P, Ayrignac X, Defebvre L, Maltete D, Martinaud O, Lefaucheur R, Guillin O, Wallon D, Chaumette B, Rondepierre P, Derache N, Fromager G, Schaeffer S, Krystkowiak P, Verny C, Jurici S, Sauvee M, Verin M, Lebouvier T, Rouaud O, Thauvin-Robinet C, Rousseau S, Rovelet-Lecrux A, Frebourg T, Campion D, Hannequin D . Phenotypic spectrum of probable and genetically-confirmed idiopathic basal ganglia calcification. Brain, 2013,1369(11):3395-3407.
[2]	Oliveira JRM, Spiteri E, Sobrido MJ, Hopfer S, Klepper J, Voit T, Gilbert J, Wszolek ZK, Calne DB, Stoessl AJ, Hutton M, Manyam BV, Boller F, Baquero M, Geschwind DH . Genetic heterogeneity in familial idiopathic basal ganglia calcification (Fahr disease). Neurology, 2004,63(11):2165-2167. doi: 10.1212/01.wnl.0000145601.88274.88 pmid: 15596772
[3]	Wang C, Li YL, Shi L, Ren J, Patti M, Wang T, de Oliveira JRm, Sobrido MJ, Quintáns B, Baquero M, Cui XN, Zhang XY, Wang LQ, Xu HB, Wang JH, Yao J, Dai XH, Liu J, Zhang L, Ma HY, Gao Y, Ma XX, Feng SL, Liu MG, Wang QK, Forster IC, Zhang X, Liu JY. Mutations in SLC20A2 link familial idiopathic basal ganglia calcification with phosphate homeostasis. Nat Genet, 2012,44(3):254-256. pmid: 22327515
[4]	Nicolas G, Pottier C, Maltete D, Coutant S, Rovelet- Lecrux A, Legallic S, Rousseau S, Vaschalde Y, Guyant- Marechal L, Augustin J, Martinaud O, Defebvre L, Krystkowiak P, Pariente J, Clanet M, Labauge P, Ayrignac X, Lefaucheur R, Le Ber I, Frébourg T, Hannequin D, Campion D . Mutation of the PDGFRB gene as a cause of idiopathic basal ganglia calcification. Neurology, 2013,80(2):181-187. doi: 10.1212/WNL.0b013e31827ccf34
[5]	Keller A, Westenberger A, Sobrido MJ, Garcia-Murias M, Domingo A, Sears RL, Lemos RR, Ordoñez-Ugalde A, Nicolas G, da Cunha JE, Rushing EJ, Hugelshofer M, Wurnig MC, Kaech A, Reimann R, Lohmann K, Dobričić V, Carracedo A, Petrović I, Miyasaki JM, Abakumova I, Mäe MA, Raschperger E, Zatz M, Zschiedrich K, Klepper J, Spiteri E, Prieto JM, Navas I, Preuss M, Dering C, Janković M, Paucar M, Svenningsson P, Saliminejad K, Khorshid HR, Novaković I, Aguzzi A, Boss A, Le Ber I, Defer G, Hannequin D, Kostić VS, Campion D, Geschwind DH, Coppola G, Betsholtz C, Klein C, Oliveira JR. Mutations in the gene encoding PDGF-B cause brain calcifications in humans and mice. Nat Genet, 2013,45(9):1077-1082. doi: 10.1038/ng.2723 pmid: 23913003
[6]	Legati A, Giovannini D, Nicolas G, López-Sánchez U, Quintáns B, Oliveira JR, Sears RL, Ramos EM, Spiteri E, Sobrido MJ, Carracedo A, Castro-Fernández C, Cubizolle S, Fogel BL, Goizet C, Jen JC, Kirdlarp S, Lang AE, Miedzybrodzka Z, Mitarnun W, Paucar M, Paulson H, Pariente J, Richard AC, Salins NS, Simpson SA, Striano P, Svenningsson P, Tison F, Unni VK, Vanakker O, Wessels MW, Wetchaphanphesat S, Yang M, Boller F, Campion D, Hannequin D, Sitbon M, Geschwind DH, Battini JL, Coppola G . Mutations in XPR1 cause primary familial brain calcification associated with altered phosphate export. Nat Genet, 2015,47(6):579-581. doi: 10.1038/ng.3289 pmid: 25938945
[7]	Yao XP, Cheng XW, Wang C, Zhao M, Guo XX, Su HZ, Lai LL, Zou XH, Chen XJ, Zhao YY, Dong EL, Lu YQ, Wu S, Li XJ, Fan GF, Yu HJ, Xu JF, Wang N, Xiong ZQ, Chen WJ. Biallelic mutations in MYORG cause autosomal recessive primary familial brain calcification. Neuron, 2018, 98(6): 1116-1123.e5. doi: 10.1016/j.neuron.2018.05.037 pmid: 29910000
[8]	Cen ZD, Chen Y, Chen S, Wang H, Yang DH, Zhang HM, Wu HW, Wang LB, Tang SY, Ye J, Shen J, Wang HT, Fu F, Chen XH, Xie F, Liu P, Xu X, Cao JZ, Cai P, Pan QQ, Li JY, Yang W, Shan PF, Li YZ, Liu JY, Zhang BR, Luo W . Biallelic loss-of-function mutations in JAM2 cause primary familial brain calcification. Brain, 2020,143(2):491-502. doi: 10.1093/brain/awz392 pmid: 31851307
[9]	Lemos RR, Ramos EM, Legati A, Nicolas G, Jenkinson EM, Livingston JH, Crow YJ, Campion D, Coppola G, Oliveira JRM . Update and mutational analysis of SLC20A2: a major cause of primary familial brain calcification. Hum Mutat, 2015,36(5):489-495. doi: 10.1002/humu.22778 pmid: 25726928
[10]	Yamada M, Tanaka M, Takagi M, Kobayashi S, Taguchi Y, Takashima S, Tanaka K, Touge T, Hatsuta H, Murayama S, Hayashi Y, Kaneko M, Ishiura H, Mitsui J, Atsuta N, Sobue G, Shimozawa N, Inuzuka T, Tsuji S, Hozumi I . Evaluation of SLC20A2 mutations that cause idiopathic basal ganglia calcification in Japan. Neurology, 2014,82(8):705-712. doi: 10.1212/WNL.0000000000000143
[11]	Jensen N, Schrøder HD, Hejbøl EK, Füchtbauer EM, de Oliveira JR, Pedersen L. Loss of function of Slc20a2 associated with familial idiopathic basal ganglia calcification in humans causes brain calcifications in mice. J Mol Neurosci, 2013,51(3):994-999. doi: 10.1007/s12031-013-0085-6
[12]	Wallingford MC, Gammill HS, Giachelli CM . Slc20a2 deficiency results in fetal growth restriction and placental calcification associated with thickened basement membranes and novel CD13 and lamininα1 expressing cells. Reprod Biol, 2016,16(1):13-26. doi: 10.1016/j.repbio.2015.12.004 pmid: 26952749
[13]	Platt RJ, Chen SD, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE, Parnas O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S, Heidenreich M, Xavier RJ, Langer R, Anderson DG, Hacohen N, Regev A, Feng GP, Sharp PA, Zhang F . CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell, 2014,159(2):440-455. doi: 10.1016/j.cell.2014.09.014
[14]	Wallingford MC, Chia JJ, Leaf EM, Borgeia S, Chavkin NW, Sawangmake C, Marro K, Cox TC, Speer MY, Giachelli CM . SLC20A2 deficiency in mice leads to elevated phosphate levels in cerbrospinal fluid and glymphatic pathway-associated arteriolar calcification, and recapitulates human idiopathic basal ganglia calcification. Brain Pathol, 2017,27(1):64-76. doi: 10.1111/bpa.12362 pmid: 26822507
[15]	Bezerra DP, Oliveira JRM . New studies on knockout mouse for the SLC20A2 gene show much more than brain calcifications. J Mol Neurosci, 2016,59(4):565-566. doi: 10.1007/s12031-016-0778-8 pmid: 27380911
[16]	Forster I, Hernando N, Sorribas V, Werner A . Phosphate transporters in renal, gastrointestinal, and other tissues. Adv Chronic Kidney Dis, 2011,18(2):63-76. doi: 10.1053/j.ackd.2011.01.006 pmid: 21406290
[17]	Villa-Bellosta R, Ravera S, Sorribas V, Stange G, Levi M, Murer H, Biber J, Forster IC . The Na+-Pi cotransporter PiT-2 ( SLC20A2) is expressed in the apical membrane of rat renal proximal tubules and regulated by dietary Pi. Am J Physiol Renal Physiol, 2009,296(4):F691-699. doi: 10.1152/ajprenal.90623.2008 pmid: 19073637
[18]	Villa-Bellosta R, Sorribas V . Compensatory regulation of the sodium/phosphate cotransporters NaPi-IIc ( SCL34A3) and Pit-2(SLC20A2) during Pi deprivation and acidosis. Pflugers Arch, 2010,459(3):499-508. doi: 10.1007/s00424-009-0746-z pmid: 19841935
[19]	Wang JP, Zhang YM . The application of Red/ET recombination to high efficient gene-targeting vector construction. Hereditas (Beijing), 2005,27(6):953-958.
	王军平, 张友明 . Red/ET重组在基因打靶载体快速构建中的应用. 遗传, 2005,27(6):953-958.
[20]	He XB, Gu F . Genome-editing: focus on the off-target effects. Chin J Biotechnol, 2017,33(10):1757-1775.
	何秀斌, 谷峰 . 基因组编辑脱靶研究进展. 生物工程学报, 2017,33(10):1757-1775.
[21]	Jensen N, Schrøder HD, Hejbøl EK, Thomsen JS, Brüel A, Larsen FT, Vinding MC, Orlowski D, Füchtbauer EM, Oliveira JRM, Pedersen L . Mice knocked out for the primary brain calcification-associated gene Slc20a2 show unimpaired prenatal survival but retarded growth and nodules in the brain that grow and calcify over time. Am J Pathol, 2018,188(8):1865-1881. doi: 10.1016/j.ajpath.2018.04.010 pmid: 29803831

Metrics

Comments

www.chinagene.cn
备案号：京ICP备09063187号

引物名称	碱基序列(5?→3?)
sgRNA1	F:caccGCTCGTGGCGATTGGCCCGAA
sgRNA1	R:aaacTTCGGGCCAATCGCCACGAGC
sgRNA2	F:caccGGCTTCTCACTCGTGGCGAT
sgRNA2	R:aaacATCGCCACGAGTGAGAAGCC
sgRNA3	F:caccGTCGGGGACTCACTGCATCGT
sgRNA3	R:aaacACGATGCAGTGAGTCCCCGAC

Construction of a striatum-specific Slc20a2 gene knockout mice model by CRISPR/Cas9 AAV system

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 6

References 21

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	Bingzheng Wang, Chao Zhang, Jiali Zhang, Jin Sun. Conditional editing of the Drosophila melanogaster genome using single transcripts expressing Cas9 and sgRNA [J]. Hereditas(Beijing), 2023, 45(7): 593-601.
[2]	Lan Wang, Fan Zeng, Rongfeng Huang, Shu Lin, Zhihui Zhang, Min-Dian Li. Adipocyte reconstitution of Npy4r gene in Npy4r silenced mice promotes diet-induced obesity [J]. Hereditas(Beijing), 2023, 45(2): 144-155.
[3]	Meizhen Liu, Liren Wang, Yongmei Li, Xueyun Ma, Honghui Han, Dali Li. Generation of genetically modified rat models via the CRISPR/Cas9 technology [J]. Hereditas(Beijing), 2023, 45(1): 78-87.
[4]	Xiaojun Zhang, Kun Xu, Juncen Shen, Lu Mu, Hongrun Qian, Jieyu Cui, Baoxia Ma, Zhilong Chen, Zhiying Zhang, Zehui Wei. A CRISPR/Cas9-Gal4BD donor adapting system for enhancing homology-directed repair [J]. Hereditas(Beijing), 2022, 44(8): 708-719.
[5]	Chong Zhang, Zixuan Wei, Min Wang, Yaosheng Chen, Zuyong He. Editing MC1R in human melanoma cells by CRISPR/Cas9 and functional analysis [J]. Hereditas(Beijing), 2022, 44(7): 581-590.
[6]	Yao Liu, Xianhui Zhou, Shuhong Huang, Xiaolong Wang. Prime editing: a search and replace tool with versatile base changes [J]. Hereditas(Beijing), 2022, 44(11): 993-1008.
[7]	Yuting Han, Bowen Xu, Yutong Li, Xinyi Lu, Xizhi Dong, Yuhao Qiu, Qinyun Che, Ruibao Zhu, Li Zheng, Xiaochen Li, Xu Si, Jianquan Ni. The cutting edge of gene regulation approaches in model organism Drosophila [J]. Hereditas(Beijing), 2022, 44(1): 3-14.
[8]	Guangwu Yang, Yuan Tian. The F-box gene Ppa promotes lipid storage in Drosophila [J]. Hereditas(Beijing), 2021, 43(6): 615-622.
[9]	Dingwei Peng, Ruiqiang Li, Wu Zeng, Min Wang, Xuan Shi, Jianhua Zeng, Xiaohong Liu, Yaoshen Chen, Zuyong He. Editing the cystine knot motif of MSTN enhances muscle development of Liang Guang Small Spotted pigs [J]. Hereditas(Beijing), 2021, 43(3): 261-270.
[10]	Na Wang, Zhilian Jia, Qiang Wu. RFX5 regulates gene expression of the Pcdhα cluster [J]. Hereditas(Beijing), 2020, 42(8): 760-774.
[11]	Guoling Li, Shanxin Yang, Zhenfang Wu, Xianwei Zhang. Recent developments in enhancing the efficiency of CRISPR/Cas9- mediated knock-in in animals [J]. Hereditas(Beijing), 2020, 42(7): 641-656.
[12]	Yingnan Chen, Jing Lu. Application of CRISPR/Cas9 mediated gene editing in trees [J]. Hereditas(Beijing), 2020, 42(7): 657-668.
[13]	Siyuan Liu, Guoqiang Yi, Zhonglin Tang, Bin Chen. Progress on genome-wide CRISPR/Cas9 screening for functional genes and regulatory elements [J]. Hereditas(Beijing), 2020, 42(5): 435-443.
[14]	Liwen Bao, Yiye Zhou, Fanyi Zeng. Advances in gene therapy for β-thalassemia and hemophilia based on the CRISPR/Cas9 technology [J]. Hereditas(Beijing), 2020, 42(10): 949-964.
[15]	Peifeng Liu, Qiang Wu. Probing 3D genome by CRISPR/Cas9 [J]. Hereditas(Beijing), 2020, 42(1): 18-31.