Chỉnh sửa gen thụ thể CD163 ở tế bào nguyên bào sợi của lợn bằng công nghệ CRISPR/CAS9

0 Người đánh giá. Xếp hạng trung bình 0

Tác giả: Khoa học Kỹ thuật Chăn nuôi

Ngôn ngữ: vie

Ký hiệu phân loại:

Thông tin xuất bản: Tạp chí Khoa học Kỹ thuật Chăn nuôi, 2023

Mô tả vật lý: tr.45696

Bộ sưu tập: Metadata

ID: 337116

Thêm vào giỏ Liên kết toàn văn

Editting CD163 recetor gene in porcine fibroblast by using CRISPR/CAS9 systemThe CD163 receptor plays an important role in the biology of porcine reproductive andrespiratory syndrome virus (PRRSV) by allowing the virus to enter and infect target cells. Thisstudy was carried out to edit the exon7- CD163 of pig fibroblasts to create a pig resistant to pathogenicporcine reproductive and respiratory syndrome virus. The CRISPR pX459 vector was attached a gRNAspecific to the exon 7 of the CD163 receptor and transformed into E. coli. The vector CRISPR pX459-CD163 was successfully cloned and isolated at a high concentration (1,700 ng/µl). The CRISPRpX459-CD163 vector was transfected into porcine fibroblast cells in medium supplemented with7,5µl Lipofectamine TM 3000 and incubated for 48 hours. Use media containing the antibioticPuromycin at concentrations from 2 to 10 µg/ml to screen successfully transfected cells. As a result,a porcine fibroblast cell line was edited in exon 7 of the CD163 gene carrying C-to-T nucleotidechange, resulting in an Alanine–to-Valine substitution. The research results have opened up theprospect of applying the combination of CRISPR/CAS9 system and cloning to create pigs resistantto pathogenic porcine reproductive and respiratory syndrome virusThụ thể CD163 đóng vai trò quan trọng trong sinh học của vius gây bệnh tai xanh, tạo điềukiện cho virus xâm nhập và gây nhiễm trùng cho các tế bào đích. Nghiên cứu này được thực hiệnvới mục đích chỉnh sửa vùng exon7 gen CD163 nhằm gây bất hoạt thụ thể CD163 ở tế bào nguyênbào sợi của lợn từ đó hạn chế khả năng xâm nhập của virus gây bênh tai xanh. Vector CRISPRpX459 được gắn đoạn gARN đặc hiệu với vùng exon7 của thụ thể CD163 ở lợn và được biếnnạp vào vi khuẩn E. coli. Vector CRISPR pX459-CD163 đã được nhân dòng và tách chiết thànhcông có nồng độ cao (1.700 ng/µl) được sử dụng để chuyển vào tế bào nguyên bào sợi lợn. VectorCRISPR pX459-CD163 được chuyển vào tế bào nguyên bào sợi lợn trong môi trường bổ sung 7,5µlLipofectamineTM 3000, ủ trong 48h. Sử dụng môi trường chứa kháng sinh Puromycin nồng độ 2-10µg/ml để sàng lọc tế bào được chuyển vector thành công. Kết quả đã tạo ra dòng tế bào nguyên bàosợi lợn được chỉnh sửa vùng exon7 gen CD163 gây biến đổi C thành T và tương ứng acid amineAlanine thành Valine. Kết quả nghiên cứu đã mở ra triển vọng trong việc ứng dụng kết hợp côngnghệ CRISPR/CAS9 và nhân bản để tạo lợn kháng virus gây bệnh tai xanh

1. Đọc trực tuyến

1. Đọc trực tuyến

Tạo bộ sưu tập với mã QR