Construction and validation of sheep VASA gene knock-in vector based on CRISPR/Cas9 system / 生物工程学报
Chinese Journal of Biotechnology
; (12): 4219-4233, 2023.
Article
en Zh
| WPRIM
| ID: wpr-1008022
Biblioteca responsable:
WPRO
ABSTRACT
This study aimed to explore the expression changes of VASA gene in sheep testis development and to construct VASA gene knock-in vector to prepare for the study on the differentiation of sheep germ cells in vitro. The testicular tissues of 3-month-old (3M) and 9-month-old (9M) sheep which represent immature and mature stages, respectively, were collected. The differential expression of VASA gene was analyzed by quantitative real-time PCR (qPCR) and Western blotting, and the location of VASA gene was detected by immunohistochemistry. The sgRNA targeting the VASA gene was designed and homologous recombination vectors were constructed by PCR. Subsequently, plasmids were transferred into sheep ear fibroblasts. The VASA gene was activated in combination with CRISPR/dCas9 technology to further verify the efficiency of the vector. The results showed that the expression level of VASA gene increased significantly with the development of sheep testis (P < 0.01), and was mainly located in spermatocytes and round spermatids. The knock-in vector of VASA gene was constructed by CRISPR/Cas9 system, and the Cas9-gRNA vector and pEGFP-PGK puro-VASA vector were transfected into ear fibroblasts. After CRISPR/dCas9 system was activated, ear fibroblasts successfully expressed VASA gene. The results suggest that VASA gene plays a potential function in sheep testicular development and spermatogenesis, and the VASA gene knock-in vector can be constructed in vitro through the CRISPR/Cas9 system. Our results provided effective research tools for further research of germ cell development and differentiation.
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Índice:
WPRIM
Asunto principal:
Plásmidos
/
Ovinos
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Técnicas de Sustitución del Gen
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Sistemas CRISPR-Cas
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Células Germinativas
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ARN Guía de Sistemas CRISPR-Cas
Límite:
Animals
Idioma:
Zh
Revista:
Chinese Journal of Biotechnology
Año:
2023
Tipo del documento:
Article