ⅱ类启动子调控crispr系统中sgrna的表达实现特异性的基因突变生物学发育生物学专业论文.docxVIP

CONTENTSCoNTENTS CONTENTS CoNTENTS Abstract in Chinese ．．1 Abstract in English．．．． ．．．．．．．． ．．．．． ．．．．．．．．．．．．． ．．．．．．．． ． ．．．．．．．．．．．． ．．．． ．．．．． ．．．．．．．．． ．． ． ．．．．．III 1 Introduction ．1 1．1 CRISPR／Cas9 system ． ． ．． ． 1 1．1．1 Discovery ofCRISPR／Cas ．1 1．1．2 Structure ofCRISPR／Cas 1 1．1．3 Mechanism ofCRISPR／CaS 2 1．1．4 Classificafion ofCRISPR／Cas ．3 1．1．5 Progress ofCRISPR／Cas9 ．．4 1．1．6ApplicationofCRISPR／Cas9 ．． ．5 1．1．7 Induced regulation ofCRISPR／Cas9 system 7 1．2 Structure of miRNA-shRNA 7 1．2．1 Discovery ofRNA interference ．7 1．2．2AppearanceofmiRNA—shRNA． ．．9 1．3 Summarise ofp53 gene ． ．．10 1．4 Destination and significance ofthe study ． ． 1 l 2 MatetiaIs and Methods ．．1 3 2．1 Materials ． ． ． ．1：I 2．1．1 Plasmid 13 2．1．2 Enzyme and kits ． 13 2．1．3 Primers and sequence ． ．15 2．1．4 Equipments ． ． ． ． ．16 2．1．5 Commen reagent preparation ． ．17 2．1．6 Biological software ． ．19 2．2 Experimental methods ．．19 2．2．1 Construction ofvector backbone ．．19 2．2．2 Construction ofmiRsh-sgp53 ．24 2．2．3 Construction ofthe mkRsh-sgp53 vector from the RNA polymerase II promoters 一26 2。2．4 Cell cultu陀and transfectiort ． ．．30 2．2．5 Sort ofpositive ceils by fluorescence-activated cell sorting 32 2．2．6 Cleavage ofTTENl ．．32 2．2．7Sanger sequencing ．33 3 Results analysis．．．．．．．．． ．．．．．．，．． ．．．．． ． ．． ．． ．．．．． ．．．．．， ．．． ．．．．．． ．．．．．．．．．．．． ．．．．．．．．．．．． ．．．34 3．1 Construction ofthe miRsh-sgp53 vector from the RNA polymerase 1I promoters 34 III 万方数据 东北农业大学理学硕士学位论文3．2 东北农业大学理学硕士学位论文 3．2 SgRNA expression status ofmiRsh-sgp53 regulated by EFIa ．36 3．3Se,RNAexpressionstatusofmiRsh-sgp53 regulatedbyembryonicstemcell-specificpromoter 36 3．4 The status ofoff-target induced by miRsh-sgp53 37 4 Discussion．． ．．．．．．．。．．．。 ．．．．．．．．．。．．．．．。