Lesch-Nyhan综合征家兔模型的建立及其表型分析

doi:10.16288/j.yczz.24-012

遗传 ›› 2024, Vol. 46 ›› Issue (5): 408-420.doi: 10.16288/j.yczz.24-012

Lesch-Nyhan综合征家兔模型的建立及其表型分析

资崯¹^,²^,³(), 郑淑文¹^,²^,³(), 宁丽¹^,²^,³, 蔺紫怡¹^,²^,³, 鹿璇¹^,²^,³, 席嘉慧¹^,²^,³, 高月¹^,²^,³, 周小青¹^,²^,³, 唐成程¹^,²^,³()

1.五邑大学药学与食品工程学院，江门 529000
2.广东省医学大动物模型重点实验室，江门 529000
3.五邑大学华南生物医药大动物模型研究院，江门 529000

收稿日期:2024-01-09 修回日期:2024-03-28 出版日期:2024-05-08 发布日期:2024-05-08
通讯作者: 唐成程 E-mail:15271856007@139.com;731726362@qq.com;wyuchemtcc@126.com
作者简介:资崯，硕士研究生，专业方向：药学及基因编辑。E-mail:15271856007@139.com.
郑淑文，硕士研究生，专业方向：基因编辑。E-mail: 731726362@qq.com.
资崯和郑淑文并列第一作者。
基金资助:
广东省基础与应用基础研究基金项目(2023A1515111163)

Establishment and characterization of Lesch-Nyhan syndrome rabbit model

Yin Zi¹^,²^,³(), Shuwen Zheng¹^,²^,³(), Li Ning¹^,²^,³, Ziyi Lin¹^,²^,³, Xuan Lu¹^,²^,³, Jiahui Xi¹^,²^,³, Yue Gao¹^,²^,³, Xiaoqing Zhou¹^,²^,³, Chengcheng Tang¹^,²^,³()

1. School of Pharmacy and Food Engineering, Wuyi University, Jiangmen 529000, China
2. Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, Jiangmen 529000, China
3. South China Institute of Large Animal Models for Biomedicine, Wuyi University, Jiangmen 529000, China

Received:2024-01-09 Revised:2024-03-28 Published:2024-05-08 Online:2024-05-08
Contact: Chengcheng Tang E-mail:15271856007@139.com;731726362@qq.com;wyuchemtcc@126.com
Supported by:
Guangdong Basic and Applied Basic Research Foundation(2023A1515111163)

摘要/Abstract

摘要：

Lesch-Nyhan综合征(Lesch-Nyhan syndrome，LNS)是一种先天性的嘌呤代谢缺陷病，次黄嘌呤鸟嘌呤磷酸核糖转移酶(hypoxanthine phosphoribosyltransferase，HPRT)是其主要致病基因，临床特征表现为尿酸分泌过多、痛风、肾结石及肾脏损伤等，目前致病机制尚未被完全阐明且无有效治愈手段。动物模型在疾病致病机理研究和治疗方式探索中发挥着重要功能。本研究采用高效的CRISPR/Cas9基因编辑技术和显微注射的方式敲除家兔(Oryctolagus cuniculus)HPRT基因建立了LNS家兔模型，以期能更好的模拟该疾病的表型。首先针对兔HPRT基因第3外显子设计一条sgRNA，将体外转录的sgRNA和Cas9 mRNA共注射到兔受精卵中，注射后的胚胎移植到代孕母兔子宫中，待仔兔出生后对其基因型及表型进行鉴定与分析。共出生4只仔兔(分别编号为R1、R2、R3和R4)，测序结果显示4只仔兔都产生了不同程度的基因修饰，基因编辑效率达100%，其中R4仔兔T载体测序结果在基因编辑靶点未检测到野生型序列。通过6-硫代鸟嘌呤(6-Thioguanine，6-TG)药物测试，证明R4仔兔HPRT酶活性缺失；肾组织HE染色发现4只仔兔表现出肾小球炎细胞浸润、集合管脱落等肾脏损失。本研究成功设计了1条能够敲除家兔HPRT基因的sgRNA，并采用CRISPR/Cas9基因编辑技术和显微注射方式成功构建了HPRT基因修饰家兔，为研究LNS综合征致病机制和治疗方式提供了新的非啮齿类动物模型。

关键词: Lesch-Nyhan综合征, HPRT, 家兔

Abstract:

Lesch-Nyhan syndrome (LNS) is a congenital defect disease that results in defective purine metabolism. It is caused by pathogenic variants of the HPRT gene. Its clinical symptoms mainly include high uric acid levels, gout, and kidney stones and damage. The mechanism of LNS has not been fully elucidated, and no cure exists. Animal models have always played an important role in exploring causative mechanisms and new therapies. This study combined CRISPR/Cas9 and microinjection to knock out the HPRT gene to create an LNS rabbit model. A sgRNA targeting exon 3 of HPRT gene was designed. Subsequently, Cas9 mRNA and sgRNA were injected into rabbit zygotes, and injected embryos were transferred to the uterus. The genotype and phenotype of rabbits were analyzed after birth. Four infant rabbits (named R1, R2, R3 and R4), which showed varying levels of gene modification, were born. The gene-editing efficiency was 100%. No wild-type sequences at the target HPRT gene were detected in R4 rabbit. Next, 6-thioguanine drug testing confirmed that HPRT enzymatic activity was deficient in R4 infant rabbit. HE staining revealed kidney abnormalities in all infant rabbits. Overall, an sgRNA capable of knocking out the HPRT gene in rabbits was successfully designed, and HPRT gene-modified rabbits were successfully constructed by using CRISPR/Cas9 technology and microinjection. This study provides a new nonrodent animal model for studying LNS syndrome.

Key words: Lesch-Nyhan syndrome, HPRT, rabbit

资崯, 郑淑文, 宁丽, 蔺紫怡, 鹿璇, 席嘉慧, 高月, 周小青, 唐成程. Lesch-Nyhan综合征家兔模型的建立及其表型分析[J]. 遗传, 2024, 46(5): 408-420.

Yin Zi, Shuwen Zheng, Li Ning, Ziyi Lin, Xuan Lu, Jiahui Xi, Yue Gao, Xiaoqing Zhou, Chengcheng Tang. Establishment and characterization of Lesch-Nyhan syndrome rabbit model[J]. Hereditas(Beijing), 2024, 46(5): 408-420.

图/表 10

图1

图2

表1

表2

用于脱靶检测的Sanger PCR引物与Hi-TOM PCR引物"

脱靶名称	脱靶位点	Sanger PCR引物(5'→3')	Hi-TOM PCR引物(5'→3')
OT1	Chr.2: 42018299-42018321	GGACAGGAGAGGGCAGTAGT CGGTGGAGAACAATCCCTCC	ggagtgagtacggtgtgcTGGCATTGAAGACGCTGACT gagttggatgctggatggGGACAGGAGAGGGCAGTAGT
OT2	Chr.3: 19777027-19777049	CATCAGCACCCTCAGTCTGG AAGATGACGAAGCCCACAGG	ggagtgagtacggtgtgcAACACACTCCTGCCCTGATC gagttggatgctggatggCATCCTAGGGCCTTGCTCC
OT3	Chr.1: 116102603-116102625	TGTGATGGCAACGGCTACTT GCACTCTTGTACTGTCAGCCT	ggagtgagtacggtgtgcTGTGATGGCAACGGCTACTT gagttggatgctggatggTGTTTTGCTGTGTTTGGGCC
OT4	Chr.18: 43811495-43811517	TGCTGTGACCAGTGATTGCT TGGCAGTTTGTCTTTGAGT	ggagtgagtacggtgtgcCTCAAGGTGGTACCCTGCTG gagttggatgctggatggACCGTACATTGTCAGACCAAAC
OT5	Chr.Un0036: 2030416-2324157	AGGAGCTGCACTGTTTGGAA GGATTGACAGTTGCAGTGCC	ggagtgagtacggtgtgcGCTCAAGGCAGTTGGGATTG gagttggatgctggatggTGAGGGTACTTCAGAAAGCTCA
OT6	Chr.9: 86296418-86296440	ACAGAAAAGCCCTGGGTCTG AAAGTCTCTTGCCAGCTGGG	ggagtgagtacggtgtgcAGGGTTGAACCAGGAGCATT gagttggatgctggatggTGGACAAAGATGCAGACTGGT
OT7	Chr.13: 27899686-27899708	GGGCTACACTTTTATGCATTTCT ACTGTTTCATAGGATCCTTCTGTAA	ggagtgagtacggtgtgcACTGTTTCATAGGATCCTTCTGTAA gagttggatgctggatggGGGCTACACTTTTATGCATTTCT
OT8	Chr.4: 75529534-75529556	GGTCTTCACCAAGCGTACCA TGGTATTGTGAGCAGCTGGG	ggagtgagtacggtgtgcTGGTATTGTGAGCAGCTGGG gagttggatgctggatggTCGTGGGGGTAGGGAGATAG
OT9	Chr.13: 142724785-142724807	TCCAAATGCAGAAGTGAGGCT TCCTGTGTGTGTGCTCTTAGTCAC	ggagtgagtacggtgtgcCCATCTCCAGTTCAGCTCCC gagttggatgctggatggCCAGTGGCTGAAGGATGGTT
OT10	Chr.Un0063: 29124-29146	TGGGGAGGAGGACTTCAGG CACGCACCCAGGGTCTCC	ggagtgagtacggtgtgcCACGCACCCAGGGTCTCC gagttggatgctggatggTGGGGAGGAGGACTTCAGG
OT11	Chr.17: 17424250-17424272	CTATTGGGAGGTCATGGGGG TCCGGGAGTATGTATTGGAGC	ggagtgagtacggtgtgcCAGGGTGGCACTATTGGGAG gagttggatgctggatggCCCACAGTTTCGGAGGTTCA
OT12	Chr.20: 13635785-13635807	CAGGCCTGTGTTTCCCAGAT TCACCTGTCGAAACCCAGTG	ggagtgagtacggtgtgcTCACCTGTCGAAACCCAGTG gagttggatgctggatggCAGGCCTGTGTTTCCCAGAT
OT13	Chr.3: 40210311-40210333	AGCCTGTTAAGCTTCGACCC TCTGATTGGCACGTTGACCC	ggagtgagtacggtgtgcTCTGATTGGCACGTTGACCC gagttggatgctggatggAGCCTGTTAAGCTTCGACCC

表2

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图4

图5

图6

图7

图8

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引物名称	引物序列(5'→3')	用途
1F	ataGCTTGCTCGAGATGTGATGAgt	构建家兔HPRT基因sgRNA
1R	taaaacGCTTGCTCGAGATGTGATGA	构建家兔HPRT基因sgRNA
T7-cr fwd	GAAATTAATACGACTCACTATA	构建制备sgRNA PCR体外转录模板
tracr rev	AAAAAAAGCACCGACTCGGTGCCAC	构建制备sgRNA PCR体外转录模板
HPRTF1	CACACTTCATGGGCACAAAGGA	扩增打靶位点
HPRTR1	CCGCCTCTCTCTCTGTGAAC	扩增打靶位点
H-HPRT-F1	ggagtgagtacggtgtgcGGAACTTTGTGTTTAATTCC	用于基因型检测的Hi-TOM PCR引物
H-HPRT-R1	gagttggatgctggatggCCGCCTCTCTCTCTGTGAAC	用于基因型检测的Hi-TOM PCR引物

Lesch-Nyhan综合征家兔模型的建立及其表型分析

Establishment and characterization of Lesch-Nyhan syndrome rabbit model

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