摘要
目的:采用CRISPR/Cas9系统建立胰岛素样生长因子受体2(IGF-ⅡR)基因定点整合的SKBR3细胞系,为探究IGF-ⅡR对人表皮生长因子受体2(HER-2)阳性乳腺癌细胞曲妥珠单抗耐药性的影响机制提供细胞模型。方法:根据AAVS1基因序列设计并合成6对小向导RNA(sgRNA),用UCA CRISPR/Cas9快速构建及活性检测试剂盒分别与pCS载体连接,筛选效率最高的sgRNA构建Cas9/sgRNA表达载体;用Asi SⅠ+Bstz17Ⅰ酶切将IGF-ⅡR片段连入hAAVS1-KI载体,构建IGF-ⅡR打靶载体,Eco RⅠ+ScaⅠ、NcoⅠ、BglⅡ酶切鉴定并测序验证;将Cas9/sgRNA载体和IGF-ⅡR打靶载体电转SKBR3细胞,经嘌呤霉素筛选,PCR鉴定,获得IGF-ⅡR基因定点整合的混合克隆细胞系;采用半固体及有限稀释方式制备单克隆。结果:构建了作用于AAVS1位点靶序列的Cas9/sgRNA载体,sgRNA活性检测显示sgRNA2具有最高效率;构建了作用于AAVS1位点靶序列的Cas9/sgRNA载体,sgRNA活性检测显示sgRNA2较sgRNA1、sgRNA3-6优势显著,具有最高效率;酶切鉴定并测序确认IGF-ⅡR打靶载体构建成功;电转染最佳条件为1200 V、20 ms、2个脉冲;嘌呤霉素最佳筛选浓度为0.5μg/mL;IGF-ⅡR打靶载体及pCS-sgRNA2质粒成功转染HER-2阳性乳腺癌细胞SKBR3,PCR鉴定及测序验证混合克隆片段基因型正确。结论:获得IGF-ⅡR基因在AAVS1位点定点整合的混合克隆细胞系,为进一步探究IGF-ⅡR对HER-2阳性乳腺癌细胞曲妥珠单抗耐药性影响的机制奠定了基础。
Objective:We developed a CRISPR/Cas9 system to establish a SKBR3 cell line with a site-integrated insulin-like growth factor receptor 2(IGF-ⅡR) gene to investigate the role of IGF-ⅡR in human epidermal growth factor receptor-2(HER-2)-positive breast cancer cells.Methods:Design and synthesize 6 pairs of small guide RNAs(sgRNAs) based on AAVS1 gene sequence, use UCA CRISPR/Cas9 rapid construction and activity detection kit to connect with pCS vector respectively, and select the most efficient sgRNA to construct Cas9/sgRNA expression vector, use Asi SⅠ+Bstz17Ⅰ to cut IGF-ⅡR. The fragment was ligated into the hAAVS1-KI vector to construct an IGF-ⅡR targeting vector, which was digested with Eco RⅠ+ScaⅠ, NcoⅠ, and BglⅡ, and then verified by sequencing. Cas9/sgRNA vector and IGF-ⅡR targeting vector were electrotransformed into SKBR3 cells and screened by puromycin, PCR identification, to obtain a mixed clone cell line of IGF-ⅡR gene site-specific integration. Using semi-solid and limited dilution for monoclonal preparation.Results:Successfully constructed a Cas9/sgRNA vector that acts on the target sequence of the AAVS1 site. The sgRNA activity test showed that sgRNA2 has the highest efficiency. Successfully constructed a Cas9/sgRNA vector that acts on the AAVS1 site target sequence. The sgRNA activity test showed that sgRNA2 is better than sgRNA1, sgRNA3-6 has significant advantages and has the highest efficiency. IGF-ⅡR fragment digested by Asi SⅠ+Bstz17Ⅰ was ligated into hAAVS1-KI vector, and Eco RⅠ+ScaⅠ were used for dual enzyme identification and sequencing to confirm the successful construction of IGF-ⅡR targeting vector. The optimal conditions for electrotransfection were 1200 V, 20 ms, and2 pulses, the optimal screening concentration of puromycin was 0.5 μg/mL. IGF-ⅡR targeting vector and Cas9/sgRNA2 vector were successfully transfected into HER-2 positive breast cancer cells SKBR3. PCR identification and sequencing confirmed that the genotypes of mixed cloned fragments were correct.Conclusion:Mixed clonal cells with the IGF-ⅡR gene integrated at the AAVS1 site were successfully established, laying a foundation for further studies on the molecular mechanism underlying the effects of IGF-ⅡR on herceptin resistance in HER-2-positive breast cancer cells.
作者
马新宇
王潇
曹中伟
MA Xin-Yu;WANG Xiao;CAO Zhong-Wei(Surgical Department of Thyroid Gland,Mammary Gland and HerniaInner Mongolia People's Hospital,Hohhot 010010,China;Research Department,Inner Mongolia People's Hospital,Hohhot 010010,China)
出处
《生物技术通讯》
CAS
2020年第3期297-304,共8页
Letters in Biotechnology
基金
内蒙古自治区科技计划(201802116)。
