[发明专利]特异性高表达棕榈酰化缺失MTDH的MDA-MB-231细胞株及构建方法

摘要

本发明涉及一种敲除野生型MTDH后重新高表达棕榈酰化缺失MTDH的MDA‑MB‑231细胞株及其构建方法。利用CRIPR/Cas9系统将乳腺癌细胞系MDA‑MB‑231中内源表达的MTDH进行敲除，之后通过定点突变的方式构建MTDH中棕榈酰化位点和sgRNA识别序列处Protospacer Adjacent Motif(PAM)位点同时突变的MTDH表达载体，借助慢病毒体系和抗性筛选获得特异性高表达棕榈酰化缺失MTDH的MDA‑MB‑231细胞株。经测序和Western Blot检测细胞株中MTDH突变及高表达，最终成功构建内源MTDH敲除而棕榈酰化缺失MTDH特异性高表达的细胞株，可作为研究MTDH棕榈酰化修饰在乳腺癌发生发展中的分子作用机制及所参与代谢调控过程的理想细胞株。

主权项

1.特异性高表达棕榈酰化缺失MTDH的MDA‑MB‑231细胞株的构建方法，是一种内源MTDH敲除而棕榈酰化位点突变MTDH特异性高表达的MDA‑MB‑231细胞株的构建方法，其特征在于：以乳腺癌细胞MDA‑MB‑231细胞作为宿主细胞通过基因敲除(CRISPR/Cas9)和突变基因过表达两步筛选实现敲除野生型MTDH而回补突变型MTDH表达；双突变后MTDH基因序列如下：ATGGCTGCACGGAGCTGGCAGGACGAGCTGGCCCAGCAGGCCGAGGAGGGCTCGGCCCGGCTGCGGGAAATGCTCTCGGTCGGCCTACC(PAM位点突变后序列，原为GG)CTTTCTGCGCACCGAGCTGGGCCTCGACCTGGGGCTGGAGCCGAAACGGTACCCCGGCTGGGTGATCCTGGTGGGCACTGGCGCGCTCGGGCTGCTGCTGCTGTTTCTGCTGGGCTACGGCTGGGCCGCGGCTAGT(棕榈酰化位点突变后序列，原为TGC)GCCGGCGCCCGCAAAAAGCGGAGGAGCCCGCCCCGCAAGCGGGAGGAGGCGGCGGCCGTGCCGGCCGCGGCCCCCGACGACCTGGCCTTGCTGAAGAATCTCCGGAGCGAGGAACAGAAGAAGAAGAACCGGAAGAAACTGTCCGAGAAGCCCAAACCAAATGGGCGGACTGTTGAAGTGGCTGAGGGTGAAGCTGTTCGAACACCTCAAAGTGTAACAGCAAAGCAGCCACCAGAGATTGACAAGAAAAATGAAAAGTCAAAGAAAAATAAGAAGAAATCAAAGTCAGATGCTAAAGCAGTGCAAAACAGTTCACGCCATGATGGAAAGGAAGTTGATGAAGGAGCCTGGGAAACTAAAATTAGTCACAGAGAGAAACGACAGCAGCGTAAACGTGATAAGGTGCTGACTGATTCTGGTTCATTGGATTCAACTATCCCTGGGATAGAAAATACCATCACAGTTACCACCGAGCAACTTACAACCGCATCATTTCCTGTTGGTTCCAAGAAGAATAAAGGTGATTCTCATCTAAATGTTCAAGTTAGCAACTTTAAATCTGGAAAAGGAGATTCTACACTTCAGGTTTCTTCAGGATTGAATGAAAACCTCACTGTCAATGGAGGAGGCTGGAATGAAAAGTCTGTAAAACTCTCCTCACAGATCAGTGCAGGTGAGGAGAAGTGGAACTCCGTTTCACCTGCTTCTGCAGGAAAGAGGAAAACTGAGCCATCTGCCTGGAGTCAAGACACTGGAGATGCTAATACAAATGGAAAAGACTGGGGAAGGAGTTGGAGTGACCGTTCAATATTTTCTGGCATTGGGTCTACTGCTGAGCCAGTTTCTCAGTCTACCACTTCTGATTATCAGTGGGATGTTAGCCGTAATCAACCCTATATCGATGATGAATGGTCTGGGTTAAATGGTCTGTCTTCTGCTGATCCCAACTCTGATTGGAATGCACCAGCAGAAGAGTGGGGCAATTGGGTAGACGAAGAAAGAGCTTCACTTCTAAAGTCCCAGGAACCAATTCCTGATGATCAAAAGGTCTCAGATGATGATAAAGAAAAGGGAGAGGGAGCTCTTCCAACTGGGAAATCCAAAAAGAAAAAAAAGAAAAAGAAGAAGCAAGGTGAAGATAACTCTACTGCACAGGACACAGAAGAATTAGAAAAAGAGATTAGAGAAGACCTTCCAGTGAATACCTCTAAAACCCGTCCAAAACAGGAAAAAGCTTTTTCCTTGAAGACCATAAGCACTAGTGATCCAGCCGAAGTACTCGTCAAAAATAGCCAGCCTATCAAGACTCTTCCACCTGCTACTTCTACCGAGCCATCTGTAATCTTATCAAAAAGTGATTCTGACAAGAGCTCTTCCCAAGTGCCGCCAATACTACAAGAGACAGATAAATCCAAGTCAAATACCAAGCAAAATAGTGTGCCTCCTTCACAGACCAAGTCTGAAACTAGCTGGGAATCTCCCAAACAAATAAAAAAGAAGAAAAAAGCCAGACGAGAAACGTGA。