利用CRISPR-Cas9系统敲除小鼠黑色素瘤细胞系MATP基因的初步研究
|
摘要
目的利用CRISPR/Cas9系统,敲除小鼠黑色素瘤细胞系的MATP基因,为MATP基因的功能研究奠定基础。方法利用http://crispr.mit.edu/网站,设计针对MATP的特异性引物,并将引物链接到pCAS9/gRNA1载体。将阳性载体转染小鼠黑色素瘤细胞系B16F10,利用无限稀释的方法获得转染后的单克隆细胞株。提取不同细胞株的基因组,通过测序的方法进一步筛选出发生MATP基因切割的细胞株,并利用Western-blot的方法验证MATP的表达情况。结果成功获得了3株MATP基因敲除的细胞株,Western-blot结果表明,该细胞株不表达MATP蛋白。结论利用pCAS9/gRNA1载体,可以实现B16F10细胞系MATP基因的敲除。
Objective To knockout the MATP gene of mouse melanoma cell line B16F10 using CRISPR/Cas9 system,and to lay foundation for the functional study of MATP gene. Methods Specific primers of MATP were designed according to the report in http://crispr. mit. edu/website. The primers were linked to pCAS9/gRNA1 vector. Then the positive vector was transfected into mouse melanoma B16F10 cells,and monoclonal cell lines were obtained by the infinite dilution method. After the genomes of different monoclonal cell lines were extracted and sequenced,the cell lines with MATP gene cleavage were screened,and the expression of MATP in these cell lines was verified by Western-blot analysis.Results Three MATP gene knockout cell lines were successfully obtained. The western-blot results showed that the cell lines did not express MATP protein. Conclusions The knockout of MATP gene in B16F10 cell line can be successfully achieved using the pCAS9/gRNA1 vector.
Objective To knockout the MATP gene of mouse melanoma cell line B16F10 using CRISPR/Cas9 system,and to lay foundation for the functional study of MATP gene. Methods Specific primers of MATP were designed according to the report in http://crispr. mit. edu/website. The primers were linked to pCAS9/gRNA1 vector. Then the positive vector was transfected into mouse melanoma B16F10 cells,and monoclonal cell lines were obtained by the infinite dilution method. After the genomes of different monoclonal cell lines were extracted and sequenced,the cell lines with MATP gene cleavage were screened,and the expression of MATP in these cell lines was verified by Western-blot analysis.Results Three MATP gene knockout cell lines were successfully obtained. The western-blot results showed that the cell lines did not express MATP protein. Conclusions The knockout of MATP gene in B16F10 cell line can be successfully achieved using the pCAS9/gRNA1 vector.
引文
[1]Lekalakala PT,Khammissa RA,Kramer B,et al.Oculocutaneous albinism and squamous cell carcinoma of the skin of the head and neck in Sub-Saharan Africa[J].J Skin Cancer,2015,2015(6).Article ID 167847.
[2]Shakil M,Ullah MI,Hussain S,et al.Homozygosity mapping of a consanguineous Pakistani family affected with oculocutaneous albinism to tyrosinase gene[J].Int J Ophthalmol,2016,9(5):794-796.
[3]吴立娟,彭晓霞,梁小云,等.眼皮肤白化病遗传基因研究进展[J].广州医学院学报,2006,34(4):58-61.
[4]Kamaraj B,Purohit R.Mutational analysis on membrane associated transporter protein(MATP)and their structural consequences in oculocutaeous albinism type 4(OCA4)—A molecular dynamics approach[J].J Cell Biochem,2016,117(11):2608-2619.
[5]Ding Y,Hong L,Chen LL,et al.Recent advances in genome editing using CRISPR/Cas9[J].Front Plant Sci,2016,7:703.
[6]He Z,Proudfoot C,Whitelaw CBA,et al.Comparison of CRISPR/Cas9 and TALENs on editing an integrated EGFP gene in the genome of HEK293FT cells[J].Springerplus,2016,5(1):1-12.
[7]Ceasar SA,Rajan V,Prykhozhij SV,et al.Insert,remove or replace:A highly advanced genome editing system using CRISPR/Cas9[J].Biochim Biophys Acta(BBA)-Mol Cell Res,2016,1863(9):2333-2344.
[8]马元武,张连峰.利用CRISPR/Cas9技术实现快速、经济的大鼠条件基因敲除技术[J].中国比较医学杂志,2014,24(3):66-66.
[9]周金伟,徐绮嫔,姚婧,等.CRISPR/Cas9基因编辑技术及其在动物基因组定点修饰中的应用[J].遗传,2015,37(10):1011-1020.
[10]Baishya R,Nayak D K,Kumar D,et al.Ursolic acid loaded PLGA nanoparticles:in vitro and in vivo evaluation to explore tumor targeting ability on B16F10 melanoma cell lines[J].Pharmaceut Res,2016,33(11):2691-703.
[11]Shin DH,Kim OH,Jun HS,et al.Inhibitory effect of capsaicin on B16-F10 melanoma cell migration via the phosphatidylinositol3-kinase/Akt/Rac1 signal pathway[J].Exp Mol Med,2008,40(5):486-494.
[2]Shakil M,Ullah MI,Hussain S,et al.Homozygosity mapping of a consanguineous Pakistani family affected with oculocutaneous albinism to tyrosinase gene[J].Int J Ophthalmol,2016,9(5):794-796.
[3]吴立娟,彭晓霞,梁小云,等.眼皮肤白化病遗传基因研究进展[J].广州医学院学报,2006,34(4):58-61.
[4]Kamaraj B,Purohit R.Mutational analysis on membrane associated transporter protein(MATP)and their structural consequences in oculocutaeous albinism type 4(OCA4)—A molecular dynamics approach[J].J Cell Biochem,2016,117(11):2608-2619.
[5]Ding Y,Hong L,Chen LL,et al.Recent advances in genome editing using CRISPR/Cas9[J].Front Plant Sci,2016,7:703.
[6]He Z,Proudfoot C,Whitelaw CBA,et al.Comparison of CRISPR/Cas9 and TALENs on editing an integrated EGFP gene in the genome of HEK293FT cells[J].Springerplus,2016,5(1):1-12.
[7]Ceasar SA,Rajan V,Prykhozhij SV,et al.Insert,remove or replace:A highly advanced genome editing system using CRISPR/Cas9[J].Biochim Biophys Acta(BBA)-Mol Cell Res,2016,1863(9):2333-2344.
[8]马元武,张连峰.利用CRISPR/Cas9技术实现快速、经济的大鼠条件基因敲除技术[J].中国比较医学杂志,2014,24(3):66-66.
[9]周金伟,徐绮嫔,姚婧,等.CRISPR/Cas9基因编辑技术及其在动物基因组定点修饰中的应用[J].遗传,2015,37(10):1011-1020.
[10]Baishya R,Nayak D K,Kumar D,et al.Ursolic acid loaded PLGA nanoparticles:in vitro and in vivo evaluation to explore tumor targeting ability on B16F10 melanoma cell lines[J].Pharmaceut Res,2016,33(11):2691-703.
[11]Shin DH,Kim OH,Jun HS,et al.Inhibitory effect of capsaicin on B16-F10 melanoma cell migration via the phosphatidylinositol3-kinase/Akt/Rac1 signal pathway[J].Exp Mol Med,2008,40(5):486-494.