基于CRISPR/Cas9构建血管平滑肌细胞特异性Mrjp1基因敲入小鼠

doi:10.16431/j.cnki.1671-7236.2023.11.008

Abstract

Abstract: 【Objective】 The aim of this study was to disclose whether the introduction of major royal jelly protein 1 (MRJP1) to vascular smooth muscle cells (VSMCs) could regulate mice blood pressure in vivo.【Method】 Based on the CRISPR/Cas9 strategy, the gRNA to Hipp11 site, Cas9, and donor vector containing honeybee Mrjp1 cDNA with the upstream Sm22α promoter were co-injected to embryos to produce the Mrjp1 gene knock-in mice that Mrjp1 gene specifically expressed in VSMCs, followed by the verification of knock-in correctness.The systolic and diastolic blood pressures of different days and Media/Lumen area ratios of thoracic aorta for wild type (WT) and homozygous (Mrjp1^+/+) mice with continuous angiotensinⅡ(AngⅡ) infusion by mini-osmotic pumps implanted subcutaneously on the back were compared.【Result】 The heterozygous (Mrjp1^+/-) mice were generated by crossing F0 chimeric with WT mice.PCR amplification and sequencing, as well as Southern blotting, for homology fragments showed that Mrjp1 gene was successfully integrated into the Hipp11 site of mouse chromosome.The WT, Mrjp1^+/- and Mrjp1^+/+mice were generated through Mrjp1^+/-mating.Mrjp1 gene was able to transcript and translate in thoracic aorta.Continuously induced by AngⅡ, the blood pressure, including systolic and diastolic, of the Mrjp1^+/+ mice showed the lower trend than that of the WT.To be exact, the systolic blood pressure at the days of 3, 6, 9 and 12 were significantly different(P<0.05), and the diastolic blood pressure at the days of 3 (P<0.05) and 6 (P<0.01) were significantly different.After the treatment of AngⅡ, the Media/Lumen area ratio of thoracic aorta of Mrjp1^+/+ mice was also significantly lower than that of the WT (P<0.05). 【Conclusion】 The specific knock-in of Mrjp1 gene to VSMCs inhibited the mice blood pressure stimulated by AngⅡ in vivo.The current work would cast light on the in-depth elucidation of MRJP1 function and the precise utilization of royal jelly.

Key words: CRISPR/Cas9; mice; gene knock-in; major royal jelly protein 1; blood pressure regulation

CLC Number:

S896.3

SHA Fangfang, FAN Pei, YANG Peichang, ZHANG Lu, LI Jianke. Generation of Mrjp1 Gene Knock-in Mice with Specific Expression in Vascular Smooth Muscle Cells via CRISPR/Cas9 Strategy[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(11): 4392-4402.

References

[1] BADIMON L, CHAGAS P, CHIVA-BLANCH G.Diet and cardiovascular disease:Effects of foods and nutrients in classical and emerging cardiovascular risk factors[J].Current Medicinal Chemistry, 2019, 26(19):3639-3651.
[2] YAP C, MIEREMET A, DE VRIES C J M, et al.Six shades of vascular smooth muscle cells illuminated by KLF4(Krüppel-like factor 4)[J].Arteriosclerosis, Thrombosis, and Vascular Biology, 2021, 41(11):2693-2707.
[3] ZHAO Q, WANG L, YANG W, et al.Interactions among genetic variants from contractile pathway of vascular smooth muscle cell in essential hypertension susceptibility of Chinese Han population[J].Pharmacogenetics and Genomics, 2008, 18(6):459-466.
[4] TOUYZ R M, ALVES-LOPES R, RIOS F J, et al.Vascular smooth muscle contraction in hypertension[J].Cardiovascular Research, 2018, 114(4):529-539.
[5] FURMANIK M, CHATROU M, VAN GORP R, et al.Reactive oxygen-forming Nox5 links vascular smooth muscle cell phenotypic switching and extracellular vesicle-mediated vascular calcification[J].Circulation Research, 2020, 127(7):911-927.
[6] NOLAN B P, SENECHAL P, WAQAR S, et al.Altered insulin-like growth factor-1 and nitric oxide sensitivities in hypertension contribute to vascular hyperplasia[J].American Journal of Hypertension, 2003, 16(5):393-400.
[7] FRATINI F, CILIA G, MANCINI S, et al.Royal jelly:An ancient remedy with remarkable antibacterial properties[J].Microbiological Research, 2016, 192:130-141.
[8] ALBALAWI A E, ALTHOBAITI N A, ALRDAHE S S, et al.Antitumor activity of royal jelly and its cellular mechanisms against Ehrlich solid tumor in mice[J].BioMed Research International, 2022, 2022:7233997.
[9] KOCOT J, KIEŁCZYKOWSKA M, LUCHOWSKA-KOCOT D, et al.Antioxidant potential of propolis, bee pollen, and royal jelly:Possible medical application[J].Oxidative Medicine and Cellular Longevity, 2018, 2018:7074209.
[10] KUNUGI H, MOHAMMED ALI A.Royal jelly and its components promote healthy aging and longevity:From animal models to humans[J].International Journal of Molecular Sciences, 2019, 20(19):4662.
[11] YAMAGUCHI K, HE S, LI Z, et al.Quantification of major royal jelly protein 1 in fresh royal jelly by indirect enzyme-linked immunosorbent assay[J].Bioscience, Biotechnology, and Biochemistry, 2013, 77(6):1310-1312.
[12] KAMAKURA M.Royalactin induces queen differentiation in honeybees[J].Nature, 2011, 473(7348):478-483.
[13] MATSUI T, YUKIYOSHI A, DOI S, et al.Gastrointestinal enzyme production of bioactive peptides from royal jelly protein and their antihypertensive ability in SHR[J].Journal of Nutritional Biochemistry, 2002, 13(2):80-86.
[14] TAKAKI-DOI S, HASHIMOTO K, YAMAMURA M, et al.Antihypertensive activities of royal jelly protein hydrolysate and its fractions in spontaneously hypertensive rats[J].Acta Medica Okayama, 2009, 63(1):57-64.
[15] FAN P, HAN B, FENG M, et al.Functional and proteomic investigations reveal major royal jelly protein 1 associated with anti-hypertension activity in mouse vascular smooth muscle cells[J].Scientific Reports, 2016, 6:30230.
[16] LIU B, CUI L S, ZHOU B, et al.Monocarbonyl curcumin analog A2 potently inhibits angiogenesis by inducing ROS-dependent endothelial cell death[J].Acta Pharmacologica Sinica, 2019, 40(11):1412-1423.
[17] FAN P, CHEN Z, TIAN P, et al.miRNA biogenesis enzyme Drosha is required for vascular smooth muscle cell survival[J].PLoS One, 2013, 8(4):e60888.
[18] TORRES-RUIZ R, RODRIGUEZ-PERALES S.CRISPR-Cas9 technology:Applications and human disease modelling[J].Briefings in Functional Genomics, 2017, 16(1):4-12.
[19] 李晓娇, 何燕华, 朱新宇, 等.CRISPR/Cas9技术在猪、鸡中的应用研究进展[J].中国畜牧兽医, 2022, 49(12):4665-4673. LI X J, HE Y H, ZHU X Y, et al.Research progress on application of CRISPR/Cas9 technology in pigs and chickens[J].China Animal Husbandry & Veterinary Medicine, 2022, 49(12):4665-4673.(in Chinese)
[20] XIAO Q, GUO D, CHEN S.Application of CRISPR/Cas9-based gene editing in HIV-1/AIDS therapy[J].Frontiers in Cellular and Infection Microbiology, 2019, 9:69.
[21] BROWNING J, ROONEY M, HAMS E, et al.Highly efficient CRISPR-targeting of the murine Hipp11 intergenic region supports inducible human transgene expression[J].Molecular Biology Reports, 2020, 47(2):1491-1498.
[22] 吴曦, 周舒雅, 刘甦苏, 等.高效切割Hipp11敲入位点的sgRNA设计及活性验证[J].实验动物科学, 2015, 32(5):26-30. WU X, ZHOU S Y, LIU S S, et al.Designation and activity verification of high efficiency sgRNAs for Hipp11 locus[J].Laboratory Animal Science, 2015, 32(5):26-30.(in Chinese)
[23] GONG H, NI J, XU Z, et al.Shp2 in myocytes is essential for cardiovascular and neointima development[J].Journal of Molecular and Cellular Cardiology, 2019, 137:71-81.
[24] JAMA H A, MURALITHARAN R R, XU C, et al.Rodent models of hypertension[J].British Journal of Pharmacology, 2022, 179(5):918-937.
[25] LUBEL J S, HERATH C B, BURRELL L M, et al.Liver disease and the renin-angiotensin system:Recent discoveries and clinical implications[J].Journal of Gastroenterology and Hepatology, 2008, 23(9):1327-1338.
[26] DE MAN F S, TU L, HANDOKO M L, et al.Dysregulated renin-angiotensin-aldosterone system contributes to pulmonary arterial hypertension[J].American Journal of Respiratory and Critical Care Medicine, 2012, 186(8):780-789.
[27] ST PAUL A, CORBETT C B, OKUNE R, et al.Angiotensin Ⅱ, hypercholesterolemia, and vascular smooth muscle cells:A perfect trio for vascular pathology[J].International Journal of Molecular Sciences, 2020, 21(12):4525.
[28] POTTHOFF S A, STAMER S, GRAVE K, et al.Chronic p38 mitogen-activated protein kinase inhibition improves vascular function and remodeling in angiotensin Ⅱ-dependent hypertension[J].Journal of the Renin-Angiotensin-Aldosterone System, 2016, 17(3):1470320316653284.
[29] TAHIR R A, BASHIR A, YOUSAF M N, et al.In Silico identification of angiotensin-converting enzyme inhibitory peptides from MRJP1[J].PLoS One, 2020, 15(2):e0228265.
[30] SATO A, UNUMA H, EBINA K.Royal jelly proteins inhibit macrophage proliferation:Interactions with native-and oxidized-low density lipoprotein[J].Protein Journal, 2021, 40(5):699-708.
[31] WAN D C, MORGAN S L, SPENCLEY A L, et al.Honey bee Royalactin unlocks conserved pluripotency pathway in mammals[J].Nature Communications, 2018, 9(1):5078.

Generation of Mrjp1 Gene Knock-in Mice with Specific Expression in Vascular Smooth Muscle Cells via CRISPR/Cas9 Strategy

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	DENG Xinyi, HUANG Jiahuan, ZHAO Chengming, MA Wenao, GE Ye, CHEN Zhibao. Effect of Astaxanthin on Immune Function in Immunosuppressed Mice [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(9): 3519-3529.
[2]	WANG Hui, FENG Baoliang, XIANG Guangming, HUANG Lei, LIU Zhiguo, LI Kui, MU Yulian. Construction of iPAMs with CD163 Monoallelic Expression and Characterization of Their Mediating PRRSV Infection [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2617-2628.
[3]	ZHANG Jiaxiang, HAN Diangang, SHI Yaling, MAO Xiaoyue, ZHAO Kaiwei, DU Xuan, XIN Jige. Construction of IPEC-J2 Cell Lines with PPARγ Gene Knockout Mediated by CRISPR/Cas9 Technology [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2670-2677.
[4]	ZHANG Xiaomeng, JIAO Anhui, WANG Yuqi, FAN Nanzhu, JIN Qingguo. Effects of Rosiglitazone Supplementation on Redox Homeostasis of Oocytes in Mice [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1818-1827.
[5]	LU Hui, CAI Gaofeng, WU Caihong, QIU Shulei, YUAN Chen, CHEN Xiaolan, LIU Yun, QIN Kaili. Effect of Agrocybe aegerita Polysaccharid on Immunomodulatory of Splenic Lymphocytes in Mice [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1828-1835.
[6]	SHAO Yongbin, ZHENG Yichuang, ZHANG Jingyi, WEI Linying, TIAN Yong, SUN Guojie, LIU Yuran, LUO Yan, GU Xinli. Study on the Anti-inflammatory Effect of Leonurine on LPS-induced Endometritis in Mice [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2149-2155.
[7]	SHI Hongzhao, LIU Yang, YAO Ruizhi, LIU Peng, LI Shize. Effects of Cold Stress Rewarming on Neurobehavioral Changes in ICR Mice [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 932-941.
[8]	XU Qianqian, YANG Wanqun, WANG Yubo, WU Fei, DAI Juanjuan, QIN Mimi, ZHUANG Jinqiu, WANG Yan, SHEN Zhiqiang, LI Jichang. Study on Immunomodulatory Effect of Prepared Radix Rehmanniae Polysaccharide on Cyclophosphamide-induced Immunosuppression in Mice [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 693-703.
[9]	WANG Wei, LI Chunzhu, WANG Shasha, SANG Rui, GE Bingjie, LI Chunting, ZHAO Xin, LIU Xinman, YU Minghong, ZHANG Xuemei. Study on the Protective Mechanism of Traditional Chinese Medicine Combined with Probiotics on Diarrhea Caused by E.coli in Mice [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 779-788.
[10]	MA Xiaolan, DONG Zhen, YUE Meishan, CHANG Guanyu, ZENG Jianguo. Effect of Macleaya cordata Leaf Extract on Ulcerative Colitis in Mice [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(11): 4664-4680.
[11]	LI Jiahao, LI Chao, ZANG Changjiang, MENG Jun, LI Hongkun, JIA Yiqi, MAILIKEZHATI Aierken, TANGNAER Tuoliubieke. Effects of Gastric and Intraperitoneal Injection of Lactobacillus rhamnosus on Fecal Flora Diversity in Mice During Pregnancy [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 151-160.
[12]	YU Kai, ZHAO Yufen, WENG Yu, WANG Qin, HAO Shaoyu, YU Boyang, DU Chenguang, SUBUDENG Gerile, LI Haijun. Effects of Dihydrotestosterone on Proliferation and Expression of Anti-Müllerian Hormone of Mouse Granulosa Cells [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3036-3043.
[13]	LI Chunting, WANG Shasha, ZHAO Xin, WANG Wei, GE Bingjie, ZHANG Xuemei. Protective Effect of Traditional Chinese Medicine Combined with Probiotics on E.coli Diarrheain Mice [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3226-3234.
[14]	PAN Jianqiu, LIUFU Sui, WU Manli, JIANG Danli, OUYANG Hongjia, SHEN Xu, XU Danning, HE Jianhua, HUANG Yunmao. Effects of Exogenous MLT and RFRP-3 on Growth and Reproductive Performance of Male Mice at the Puberty [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(7): 2622-2630.
[15]	MENG Jiejie, SONG Yue, FAN Wenjie, YANG Le, XING Jiayou, WANG Jiang, CHU Beibei, YANG Guoyu, WANG Mengdi. Effect of Toll-like Receptor 7 Gene Knockout on Proliferation of Vesicular Stomatitis Virus [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(6): 2011-2021.