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薛璐, 歐陽聰, 孫寧遠, 秦緒慧.基于CRISPR/Cas9技術建立敲除Plac9基因的人胚腎細胞株[J].中南民族大學學報自然科學版,2019,(2):189-192
基于CRISPR/Cas9技術建立敲除Plac9基因的人胚腎細胞株
Establishment of Plac9 Gene Knockout 293T Cell Line via CRISPR/Cas9 Technique
  
DOI:10.12130/znmdzk.20190207
中文關鍵詞: CRISPR/Cas9  基因編輯技術  基因敲除  Plac9
英文關鍵詞: CRISPR/Cas9  genome editing technique  gene knockout  Plac9
基金項目:國家自然科學基金資助項目 (31101047)
作者單位
薛璐, 歐陽聰, 孫寧遠, 秦緒慧 中南民族大學 生命科學學院,生物醫學研究所,武陵山區特色資源植物種質保護與利用湖北省重點實驗室,武漢430074 
摘要點擊次數: 191
全文下載次數: 181
中文摘要:
      目的:通過CRISPR/Cas9基因編輯技術和流式細胞術相結合,獲得可編輯Plac9基因敲除的人胚腎細胞株(293T).方法:根據Plac9外顯子設計了4個sgRNAs(S1,S2,S3,S4),分別將其與PX458載體連接,構建了PX458-S1(S2/S3/S4)載體.通過轉染試劑lipo2000將表達載體分別轉染至293T細胞中,并以流式細胞術對帶綠色熒光蛋白標記的細胞進行單細胞分選,分選后的細胞培養一段時間后,用基因組測序和錯配酶酶切進行篩選.從篩選好的細胞中提取蛋白,進行Western Blot檢測敲除效率.結果:采用CRISPR/Cas9和流式細胞術結合技術成功構建了Plac9蛋白表達缺失的人胚腎細胞株. 結論:該方法簡便快捷、效率高,可廣泛地用于編輯各種細胞和細胞功能研究.
英文摘要:
      Objective: To obtain editable Plac9 gene knockout 293T cell line through CRISPR/Cas9 gene editing and flow cytometry. Methods: 4 sgRNAs (S1,S2,S3,S4) were designed and respectively connected with PX458 carrier, according to the exons of Plac9 gene, then vectors PX458-S1(S2/S3/S4)were constructed. 293T cells were transfected with the expression vectors by transfection reagent lipo2000. The green fluorescent protein labeled cells were then concentrated via single cell sorting by flow cytometry. Selected cells were cultured and identified with genomic sequencing and mismatched enzyme digestion. The protein was extracted from the selected cells and the knockout efficiency was measured by Western Blot. Results: Protein Plac9 knockout 293T cell line was successfully constructed by employing CRISPR/Cas9 and flow cytometry technology. Conclusion: This method is convenient and efficient, which can be widely applied for editing any type of cell lines and for the study of cell functions.
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