MSTN基因编辑牛的鉴定及基因分型新方法
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摘要
基因编辑是目前进行遗传修饰的重要手段,但因编辑时缺失的片段小而给基因编辑的鉴定和分型带来困难。该研究以肌肉生成抑制素基因(Myostation,MSTN)编辑牛为材料,建立功能核酸PCR(Functional nucleic acid PCR,FNA-PCR)方法对其进行鉴定及基因分型。针对编辑位点的两种等位基因设计两条不同的正向引物和一条共用的反向引物,其中一条正向引物是用于检测野生型等位基因,3'端终止于编辑位点,同时对它的5'端进行功能核酸接头设计;另一条正向引物用于检测编辑型等位基因,3'端跨越编辑位点向后延伸几个碱基。这种设计可使野生型和编辑型等位基因扩增产物大小不同。通过对PCR反应体系和条件的优化,建立了一种通过一次PCR反应准确对三种不同基因型进行分型的FNA-PCR新方法。检测方法灵敏度高,检测限为0.1ng。该研究中所建立的FNA-PCR方法可用于基因编辑检测及其它小片段缺失的基因分型,具有简便、准确、灵敏、成本低等优点。
Gene editing is a critical technique to generate genetically modified animals;however,it is hard to identify and genotype gene-edited animal because deleted fragment during editing is quite small. In this study,functional nucleic acid PCR(FNA-PCR)technique was established for identifying and genotyping MSTN gene-edited cattle. Based on 2 alleles for edited sites,2 different forward primers and one common reverse primer were designed. One forward primer was used to identify the allele of wild type samples,its 3'end was terminated at the edited site,and functional nucleic acid joint was designed to its 5'end. The other forward primer was used to detect MSTN gene-edited cattle in which the 3'end extended several nucleotides beyond the gene-edited site. This unique design allowed the amplified allele sizes of gene-edited and wild-type varied. By optimizing PCR reaction system and conditions,a novel FNA-PCR method,3 different genes were accurately genotyped in one PCR reaction,was established. FNA-PCR was high in sensitivity with detection limit 0.1 ng of MSTN gene-edited cattle genome DNA. In conclusion,FNA-PCR is a simple,accurate and rapid for identifying gene-edited animal and genotyping those smallfragment-deletion genes.
Gene editing is a critical technique to generate genetically modified animals;however,it is hard to identify and genotype gene-edited animal because deleted fragment during editing is quite small. In this study,functional nucleic acid PCR(FNA-PCR)technique was established for identifying and genotyping MSTN gene-edited cattle. Based on 2 alleles for edited sites,2 different forward primers and one common reverse primer were designed. One forward primer was used to identify the allele of wild type samples,its 3'end was terminated at the edited site,and functional nucleic acid joint was designed to its 5'end. The other forward primer was used to detect MSTN gene-edited cattle in which the 3'end extended several nucleotides beyond the gene-edited site. This unique design allowed the amplified allele sizes of gene-edited and wild-type varied. By optimizing PCR reaction system and conditions,a novel FNA-PCR method,3 different genes were accurately genotyped in one PCR reaction,was established. FNA-PCR was high in sensitivity with detection limit 0.1 ng of MSTN gene-edited cattle genome DNA. In conclusion,FNA-PCR is a simple,accurate and rapid for identifying gene-edited animal and genotyping those smallfragment-deletion genes.
引文
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[5]Lee S J,Mcpherron A C.Regulation of myostatin activity and muscle growth[J].Proc Natl Acad Sci USA,2001,98(16):9306-9311.
[6]Mcpherron A C,Lee S J.Double muscling in cattle due to mutations in the myostatin gene[J].Proc Natl Acad Sci USA,1997,94(23):12457-12461.
[7]Rao S,Fujimura T,Matsunari H,et al.Efficient modification of the myostatin gene in porcine somatic cells and generation of knockout piglets[J].Mol Reprod Dev,2015,83(1):61-70.
[8]Hanset R,Michaux C,Dessy-Doize C,et al.Studies on the 7th rib cut in double muscled and conventional cattle.Anatomical,Histological and Biochemical Aspects[M]//Muscle Hypertrophy of Genetic Origin and its use to Improve Beef Production.Springer Netherlands,1982:341-349.
[9]Shan Q,Wang Y,Li J,et al.Genome editing in rice and wheat using the CRISPR/Cas system[J].Nat Protoc,2014,9(10):2395.
[10]Mashal R D,Koontz J,Sklar J.Detection of mutations by cleavage of DNA heteroduplexes with bacteriophage resolvases[J].Nature Genetics,1995,9(2):177-83.
[11]Wittwer CT,Reed GH,Gundry CN,et al.High-resolution genotyping by amplicon melting analysis using LCGreen[J].Clin Chem,2003,49(6):853-860.
[12]Stoltenburg R,Reinemann C,Strehlitz B.SELEX-A(r)evolutionary method to generate high-affinity nucleic acid ligands[J].Biomol Eng,2007,24(4):381-403.
[13]Silverman SK.Catalytic DNA:scope,applications,and biochemistry of deoxyribozymes[J].Trends in Biochemical Sciences,2016,41(7):595-609.
[14]Sambrook J,Fritsch EF,Maniatis T.Molecular cloning:a laboratory manual[M].2nd ed.New York:Cold Spring Harbor Laboratory Press,1989.
[15]Yang P,Wang J,Gong G,et al.Cattle mammary bioreactor generated by a novel procedure of transgenic cloning for large-Scale production of functional human lactoferrin[J].PLoS One,2008,3(10):e3453.
[16]Maga E A,Cullor J S,Smith W,et al.Human lysozyme expressed in the mammary gland of transgenic dairy goats can inhibit the growth of bacteria that cause mastitis and the cold-spoilage of milk[J].Foodborne Pathogens&Disease,2006,3(4):384.
[17]Wang J,Yang P,et al.Expression and characterization of bioactive recombinant human alpha-lactalbumin in the milk of transgenic cloned cows[J].J Dairy Sci,2008,91(12):4466-4476.
[18]Hua Y,Wang C,Huang J,et al.A simple and efficient method for CRISPR/Cas9-induced mutant screening[J].Journal of Genetics and Genomics,2017,44(4):207-213
[19]Vouillot L,Thélie A,Pollet N.Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by Engineered Nucleases[J].G3(Bethesda),2015,5(3):407-415.
[20]Thomas HR,Percival SM,Yoder BK,et al.High-throughput genome editing and phenotyping facilitated by high resolution melting curve analysis[J].PLoS One,2014,9(12):e114632.