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

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

• Research Article • Previous Articles Next Articles

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 Online:2024-05-08 Published: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)

Abstract

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

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.

Figures/Tables 10

Fig. 1

Fig. 2

Table 1

Table 2

Sanger PCR and Hi-TOM PCR primer for off-target detection"

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

Table 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 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

Establishment and characterization of Lesch-Nyhan syndrome rabbit model

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