第十一章 动物基因工程

第十一章动物基因工程第一节动物细胞基因工程Animal cells are advantageous for the production of recombinant animal proteins because they perform authentic post-translational modifications.Cell cultures have been used on a commercial scale to synthesize products一、动物细胞Primary cellsUndergo only a few divisions before undergoing senescence.Secondary cell line: (10–20 times)Immortal cell lineHeLacells、、Chinese hamster ovary (CHO) cellsTransformation:The changes that cells undergo unregulated cell division and growth resulting from a viral infection or other change within the cellTransfection:In higher eukaryotes the process of transferring foreign DNA into cells is called transfection.二、动物细胞的转染Transient transfection.The foreign DNA is not integrated into the genome and does not contain an origin of replication Be degraded or diluted during cell division.A rapid way to analyseforeign genes and gene products within cells.Stabletransfection.The foreign DNA becomes integrated into an apparently randomlocation within the genome1.Chemical TransfectionThe efficiency of naked DNA transfection can be greatly improved if the DNA is precipitated in the presence of the cells.DNA/calcium phosphate coprecipitate method将待转染的DNA同CaCl2混合制成CaCl2-DNA溶液逐滴加入到Hepes-磷酸盐缓冲液中，形成磷酸钙-DNA共沉淀用吸管将共沉淀物加到培养的哺乳动物单层细胞表面，细胞通过吞噬作用捕获DNA，频率约10%部分DNA可进入细胞核，瞬时表达或整合进入基因组2 Electroporation3 Liposome-mediated Transfection脂质体(liposomes)是一种人造的脂质小泡，外周是脂双层，内部是水腔4 DNA Binding PeptidesA number of peptide sequences have been shown to be able to bind to, and condense, DNA to make it more amenable for entry into cellsThe tetrapeptide: serine–proline–lysine–lysine5. Direct DNA TransferMicroinjection, especially useful for large cells.Integration of injected foreign DNA occurs at a very low frequency, but the expression of the foreign gene can be followed for many cell generations.三、病毒载体动物病毒含有可被真核细胞识别的启动子有许多病毒在感染周期中持续复制，达到很高的拷贝数有些病毒可整合到宿主核基因组中病毒可高效的感染宿主细胞1. SV40病毒载体猿猴空泡病毒40 (SV40)感染猿猴细胞后，产生感染性的病毒颗粒，裂解细胞——Permissive cellSV40感染啮齿动物（仓鼠和小鼠），将病毒基因组整合到寄主的染色体上，不产生感染性颗粒——Non-Permissive celli. SV40的生活史SV40 has a circular double-stranded DNA genome of approximately 5.2 kbThe genome has two transcription units, known as the earlyand late regions, which face in opposite directions.Both transcripts produce multiple products by alternative splicing.早期基因：T和t抗原，与病毒的致瘤作用有关，并对病毒DNA复制的起始十分重要晚期基因：VP1、VP2、VP3，编码病毒的衣壳蛋白早期转录产生T抗原和t抗原，在T抗原作用下起始病毒DNA的复制，在DNA复制后，开始晚期转录，表达产生病毒的衣壳蛋白VP1、VP2、VP3，然后装配成病毒颗粒，裂解细胞ii. SV40载体野生型的SV40病毒颗粒只能包装其基因组大小的DNA用外源DNA取代病毒的早期或晚期基因，在辅助病毒的帮助下形成含有外源DNA的病毒颗粒i）SV40晚期区取代的载体用外源DNA取代SV40晚期基因区用早期基因有缺失的SV40作为辅助病毒将两种DNA共同转染猴细胞，彼此提供基因产物，最后可形成病毒颗粒，一些病毒颗粒中含有外源DNA，一些含有突变的病毒基因。

释放的病毒再感染其它的猴细胞，在这一轮感染周期中，外源基因得以高效表达ii）早期取代载体用外源DNA取代SV40早期基因区，利用晚期基因有缺失的SV40作为辅助病毒，将两种DNA 共同转染猴细胞COS细胞：将一个复制起点部位缺失的SV40基因组整合到猴细胞的染色体上所形成的细胞系。

早期基因表达可产生T和t抗原，但由于没有复制起点，SV40 DNA不能复制，也就不能产生晚期基因产物，故COS细胞可积累T抗原用早期取代载体感染cos细胞，在cos细胞所提供的T抗原的帮助下，病毒DNA可进行复制表达晚期基因，最后可包装成病毒颗粒，在病毒颗粒只有早期取代载体或其重组DNA分子iii）SV40瞬时表达载体带有SV40复制起点的质粒型表达载体该载体可在大肠杆菌细胞内复制可直接转染哺乳动物细胞不通过病毒颗粒的包装，可插入较大的DNA片段转入动物细胞不发生裂解当重组DNA分子转入COS细胞后, 在cos细胞所提供的T抗原的帮助下，能大量复制, 其拷贝数可达20-40万，并高效表达载体上的外源基因由于转染质粒的复制毫无节制地不断进行，直至细胞可能由于无法忍受它的染色体外复制如此大量的DNA而最终死亡，因而这一系统是瞬时表达系统2、腺病毒载体i. 腺病毒（Adenovirus）的特点分布广泛的呼吸道病毒(25% common colds)，无包膜(envelope)线状双链DNA，36kb，可在寄主细胞内大量增殖（105per infected cell ）在腺病毒感染周期中，寄主蛋白质合成被关闭，因而能最大限度合成病毒编码的蛋白质ii. 腺病毒载体ITR和Ψ是腺病毒基因组复制和包装必不可少的部分，除此之外的病毒基因组均可被置换，最大可插入8kb片段在辅助病毒或相应细胞系（如人293肾细胞系，提供E1)帮助下产生重组腺病毒颗粒Advantages of adenoviral vectorsHighly efficient at getting DNA into cells.Infect both replicating and differentiated cells.Do not integrate into the host genome, has no mutagenic effects caused by random integration events.DisadvantagesThe viral DNA does not integrate into the host.Adenoviral vectors are based on an extremely common human pathogen and in vivo delivery may be hampered by prior host immune response to one type of virus.3. 腺相关病毒载体Adeno-associated Virus (AAV)AAV was first discovered as a contaminant of adenovirus preparationsA member of the parvovirus familyss-DNA genome, ～5000 nucleotidesno specific human disease associated with AAVAAVsare naturally replication deficient (need helper virus)In the absence of helper virus, the AAV genome integrates into the host cell at specific location on chromosome 19.Advantage of AAVIntroduce foreign DNA into a wide variety of cell types, including neurons, muscle and epithelial cellsIntegrate into specific sitePromising vector for gene therapy4. 逆转录病毒载体Retrovirusesare the only animal viruses that integrate into the host cell genome during the normal growth cycle.There are two classes of retrovirus that infect humans–the HTLV retroviruses (e.g. human T-cell leukemia virus, HTLV-1), and the lentiviruses(e.g. HIV-1)Retroviruses have been found in a wide variety of organisms, including both vertebrates and invertebrates.i.逆转录病毒的生活史一种整合型的单链RNA病毒，病毒颗粒中一般携带有两个拷贝的单链RNA基因组 病毒envelope proteins 与宿主细胞表面特异受体结合摄入病毒颗粒逆病毒感染宿主细胞后，其RNA在体内由自身编码的逆转录酶逆转录成相应的双链DNA分子，进入细胞核，并在整合酶的作用下，随机整合到宿主染色体上逆病毒与肿瘤仅有gag, pol, env三种基因的逆病毒，不能转化细胞形成肿瘤，细胞的转化是由onc（肿瘤）基因引起的逆病毒的癌基因来自于动物细胞的原癌基因（proto-oncogene）少数逆病毒含有完整的gag, pol, env基因和额外的癌基因，如劳斯肉瘤病毒RSV大多数病毒是由癌基因取代了gag, pol, env三个基因中的一个或两个，因此这种病毒是缺陷型的，需要辅助病毒才能增殖ii. 逆病毒载体将逆病毒DNA克隆到质粒pBR322上去掉gag, pol, env三个基因的大部或全部序列，保留5’-LTR，ψ，3’-LTR插入选择标记基因①利用辅助病毒重组的逆病毒质粒载体可在大肠杆菌细胞内增殖将纯化的DNA转入动物细胞，筛选出稳定的转化子转化细胞能表达外源基因，并合成重组逆病毒的RNA分子如何将转化细胞产生的重组病毒的RNA分子包装到病毒颗粒中？将辅助病毒超感染转化细胞辅助病毒产生的外壳蛋白能包装重组病毒的RNA分子及逆转录酶，形成病毒颗粒包装的病毒颗粒感染宿主细胞，基因转移率可达100%，并且以单拷贝形式整合包装的病毒颗粒中有辅助病毒存在，影响了重组病毒的感染效率，并且辅助病毒可能致病②包装细胞系在包装细胞系的染色体上整合了一个缺失了ψ序列的逆病毒DNA；或是在其染色体的两个位点分别整合缺失了ψ序列的5’-LTR-gag-3’LTR和5’-LTR-pol-env-3’LTR区段包装细胞系能组成型表达逆病毒的全部蛋白质，但不能包装缺失了ψ序列的RNA将纯化的DNA转染包装细胞系，产生有感染能力的重组逆病毒颗粒Advantages of retroviral vectorsIntegration of the viral genome into the host allows for the long-term expression of the integrated foreign gene.a highly efficient mechanism for the transfer of DNA into cells.disadvantagesRandomly integration may have deleterious effects on the host cellInfect only dividing cells (except for lentiviruses, e.g. HIV1).第二节哺乳动物基因转移的遗传标记Endogenous selectable marker geneThese genes present in wild-type cellsThey can only be used with mutant cell lines in which the corresponding host gene is non-functional.Dominant selectable markersConfer a phenotype that is entirely novel to the cell and hence can be used in any cell type1. 胸苷激酶基因选择系统TK－细胞把细胞培养在加有胸苷类似物5-溴尿嘧啶脱氧核苷（BUdR）的培养基中，在胸苷激酶（TK）的催化下，BUdR便掺入到细胞的DNA分子上，致使DNA复制出现错误。