動(dòng)物基因工程

1、第十一章動(dòng)物基因工程第一節(jié)動(dòng)物細(xì)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、動(dòng)物細(xì)胞Primary cellsUndergo only a few divisions before undergo

2、ing senescence.Secondary cell line: (1020 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 proces

3、s of transferring foreign DNA into cells is called transfection.二、動(dòng)物細(xì)胞的轉(zhuǎn)染Transient transfection.The foreign DNA is not integrated into the genome and does not contain an origin of replicationBe degraded or diluted during cell division.A rapid way to analyseforeign genes and gene products within cell

4、s.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將待轉(zhuǎn)染的D

5、NA同CaCl2混合制成CaCl2-DNA溶液逐滴加入到Hepes-磷酸鹽緩沖液中，形成磷酸鈣-DNA共沉淀用吸管將共沉淀物加到培養(yǎng)的哺乳動(dòng)物單層細(xì)胞表面，細(xì)胞通過吞噬作用捕獲DNA，頻率約10%部分DNA可進(jìn)入細(xì)胞核，瞬時(shí)表達(dá)或整合進(jìn)入基因組2 Electroporation3 Liposome-mediated Transfection脂質(zhì)體(liposomes)是一種人造的脂質(zhì)小泡，外周是脂雙層，內(nèi)部是水腔4 DNA Binding PeptidesA number of peptide sequences have been shown to be able to bind to, a

6、nd condense, DNA to make it more amenable for entry into cellsThe tetrapeptide: serineprolinelysinelysine5. 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

7、for many cell generations.三、病毒載體動(dòng)物病毒含有可被真核細(xì)胞識(shí)別的啟動(dòng)子有許多病毒在感染周期中持續(xù)復(fù)制，達(dá)到很高的拷貝數(shù)有些病毒可整合到宿主核基因組中病毒可高效的感染宿主細(xì)胞1. SV40病毒載體猿猴空泡病毒40 (SV40)感染猿猴細(xì)胞后，產(chǎn)生感染性的病毒顆粒，裂解細(xì)胞Permissive cellSV40感染嚙齒動(dòng)物（倉鼠和小鼠），將病毒基因組整合到寄主的染色體上，不產(chǎn)生感染性顆粒Non-Permissive celli. SV40的生活史SV40 has a circular double-stranded DNA genome of approximatel

8、y 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，與病毒的致瘤作用有關(guān)，并對(duì)病毒DNA復(fù)制的起始十分重要晚期基因：VP1、VP2、VP3，編碼病毒的衣殼蛋白早期轉(zhuǎn)錄產(chǎn)生T抗原和t抗原，在T抗原作用下起始病毒DNA的復(fù)制，在DNA復(fù)制后，開始晚期轉(zhuǎn)錄，表達(dá)產(chǎn)生

9、病毒的衣殼蛋白VP1、VP2、VP3，然后裝配成病毒顆粒，裂解細(xì)胞ii. SV40載體野生型的SV40病毒顆粒只能包裝其基因組大小的DNA用外源DNA取代病毒的早期或晚期基因，在輔助病毒的幫助下形成含有外源DNA的病毒顆粒i）SV40晚期區(qū)取代的載體用外源DNA取代SV40晚期基因區(qū)用早期基因有缺失的SV40作為輔助病毒將兩種DNA共同轉(zhuǎn)染猴細(xì)胞，彼此提供基因產(chǎn)物，最后可形成病毒顆粒，一些病毒顆粒中含有外源DNA，一些含有突變的病毒基因。釋放的病毒再感染其它的猴細(xì)胞，在這一輪感染周期中，外源基因得以高效表達(dá)ii）早期取代載體用外源DNA取代SV40早期基因區(qū)，利用晚期基因有缺失的SV40作為輔

10、助病毒，將兩種DNA共同轉(zhuǎn)染猴細(xì)胞COS細(xì)胞：將一個(gè)復(fù)制起點(diǎn)部位缺失的SV40基因組整合到猴細(xì)胞的染色體上所形成的細(xì)胞系。早期基因表達(dá)可產(chǎn)生T和t抗原，但由于沒有復(fù)制起點(diǎn)，SV40 DNA不能復(fù)制，也就不能產(chǎn)生晚期基因產(chǎn)物，故COS細(xì)胞可積累T抗原用早期取代載體感染cos細(xì)胞，在cos細(xì)胞所提供的T抗原的幫助下，病毒DNA可進(jìn)行復(fù)制表達(dá)晚期基因，最后可包裝成病毒顆粒，在病毒顆粒只有早期取代載體或其重組DNA分子iii）SV40瞬時(shí)表達(dá)載體帶有SV40復(fù)制起點(diǎn)的質(zhì)粒型表達(dá)載體該載體可在大腸桿菌細(xì)胞內(nèi)復(fù)制可直接轉(zhuǎn)染哺乳動(dòng)物細(xì)胞不通過病毒顆粒的包裝，可插入較大的DNA片段轉(zhuǎn)入動(dòng)物細(xì)胞不發(fā)生裂解當(dāng)重組

11、DNA分子轉(zhuǎn)入COS細(xì)胞后, 在cos細(xì)胞所提供的T抗原的幫助下，能大量復(fù)制, 其拷貝數(shù)可達(dá)20-40萬，并高效表達(dá)載體上的外源基因由于轉(zhuǎn)染質(zhì)粒的復(fù)制毫無節(jié)制地不斷進(jìn)行，直至細(xì)胞可能由于無法忍受它的染色體外復(fù)制如此大量的DNA而最終死亡，因而這一系統(tǒng)是瞬時(shí)表達(dá)系統(tǒng)2、腺病毒載體i. 腺病毒（Adenovirus）的特點(diǎn)分布廣泛的呼吸道病毒(25% common colds)，無包膜(envelope)線狀雙鏈DNA，36kb，可在寄主細(xì)胞內(nèi)大量增殖（105per infected cell ）在腺病毒感染周期中，寄主蛋白質(zhì)合成被關(guān)閉，因而能最大限度合成病毒編碼的蛋白質(zhì)ii. 腺病毒載體ITR和

12、是腺病毒基因組復(fù)制和包裝必不可少的部分，除此之外的病毒基因組均可被置換，最大可插入8kb片段在輔助病毒或相應(yīng)細(xì)胞系（如人293腎細(xì)胞系，提供E1)幫助下產(chǎn)生重組腺病毒顆粒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 integratio

13、n 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. 腺相關(guān)病毒載體Adeno-associated Virus (AAV)AAV was first discovered as a contamin

14、ant 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 c

15、hromosome 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. 逆轉(zhuǎn)錄病毒載體Retrovirusesare the only animal viruses that integrate into the host cell genome during the norma

16、l growth cycle.There are two classes of retrovirus that infect humansthe 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. 逆轉(zhuǎn)錄病毒的生活史一種整合型的單鏈RNA病毒，病

17、毒顆粒中一般攜帶有兩個(gè)拷貝的單鏈RNA基因組􀂄病毒envelope proteins 與宿主細(xì)胞表面特異受體結(jié)合攝入病毒顆粒􀂄逆病毒感染宿主細(xì)胞后，其RNA在體內(nèi)由自身編碼的逆轉(zhuǎn)錄酶逆轉(zhuǎn)錄成相應(yīng)的雙鏈DNA分子，進(jìn)入細(xì)胞核，并在整合酶的作用下，隨機(jī)整合到宿主染色體上逆病毒與腫瘤僅有g(shù)ag, pol, env三種基因的逆病毒，不能轉(zhuǎn)化細(xì)胞形成腫瘤，細(xì)胞的轉(zhuǎn)化是由onc（腫瘤）基因引起的逆病毒的癌基因來自于動(dòng)物細(xì)胞的原癌基因（proto-oncogene）少數(shù)逆病毒含有完整的gag, pol, env基因和額外的癌基因，如勞斯肉瘤病毒RSV大多數(shù)病毒是由癌基因取

18、代了gag, pol, env三個(gè)基因中的一個(gè)或兩個(gè)，因此這種病毒是缺陷型的，需要輔助病毒才能增殖ii. 逆病毒載體將逆病毒DNA克隆到質(zhì)粒pBR322上去掉gag, pol, env三個(gè)基因的大部或全部序列，保留5-LTR，3-LTR插入選擇標(biāo)記基因 利用輔助病毒重組的逆病毒質(zhì)粒載體可在大腸桿菌細(xì)胞內(nèi)增殖將純化的DNA轉(zhuǎn)入動(dòng)物細(xì)胞，篩選出穩(wěn)定的轉(zhuǎn)化子轉(zhuǎn)化細(xì)胞能表達(dá)外源基因，并合成重組逆病毒的RNA分子如何將轉(zhuǎn)化細(xì)胞產(chǎn)生的重組病毒的RNA分子包裝到病毒顆粒中？將輔助病毒超感染轉(zhuǎn)化細(xì)胞輔助病毒產(chǎn)生的外殼蛋白能包裝重組病毒的RNA分子及逆轉(zhuǎn)錄酶，形成病毒顆粒包裝的病毒顆粒感染宿主細(xì)胞，基因轉(zhuǎn)移率可

19、達(dá)100%，并且以單拷貝形式整合包裝的病毒顆粒中有輔助病毒存在，影響了重組病毒的感染效率，并且輔助病毒可能致病包裝細(xì)胞系在包裝細(xì)胞系的染色體上整合了一個(gè)缺失了序列的逆病毒DNA；或是在其染色體的兩個(gè)位點(diǎn)分別整合缺失了序列的5-LTR-gag-3LTR和5-LTR-pol-env-3LTR區(qū)段包裝細(xì)胞系能組成型表達(dá)逆病毒的全部蛋白質(zhì)，但不能包裝缺失了序列的RNA將純化的DNA轉(zhuǎn)染包裝細(xì)胞系，產(chǎn)生有感染能力的重組逆病毒顆粒Advantages of retroviral vectorsIntegration of the viral genome into the host allows for

20、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).第二節(jié)哺乳動(dòng)物基因轉(zhuǎn)移的遺傳標(biāo)記Endogenous selectable

21、 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. 胸苷激酶基因選擇系統(tǒng)TK細(xì)胞把細(xì)胞培養(yǎng)在加有胸苷類似物5

22、-溴尿嘧啶脫氧核苷（BUdR）的培養(yǎng)基中，在胸苷激酶（TK） 的催化下， BUdR便摻入到細(xì)胞的DNA分子上，致使DNA復(fù)制出現(xiàn)錯(cuò)誤。具有BUdR-DNA 的細(xì)胞對(duì)UV特別敏感，被迅速地殺死，而少數(shù)TK的細(xì)胞便能存活下來HAT選擇法：在含有次黃嘌呤(Hypoxanthine)、氨基蝶呤(Aminopterin)和胸苷(Thymidine)的培養(yǎng)基中篩選TK+細(xì)胞（HAT blocks de novo pathway）共轉(zhuǎn)化選擇在磷酸鈣沉淀中，有兩種形體上沒有連接的DNA組成的混合物，能夠同時(shí)轉(zhuǎn)化受體細(xì)胞將無選擇標(biāo)記的DNA與具TK選擇標(biāo)記基因的DNA進(jìn)行混合轉(zhuǎn)化為了獲得共轉(zhuǎn)化的細(xì)胞，須將TK標(biāo)

23、記基因同極大超量的共轉(zhuǎn)化的DNA混合，如120000共轉(zhuǎn)化DNA先形成多連體的形式，然后共同整合到宿主細(xì)胞的基因組中，最長可達(dá)2000kb外源DNA可整合到核基因組的任何部位把TK基因先與非選擇標(biāo)記基因連接起來，也能共轉(zhuǎn)化2. 二氫葉酸還原酶基因選擇系統(tǒng)基本原理：DHFR催化二氫葉酸(DHF)還原成四氫葉酸(THF)氨基蝶呤(aminopterin)和氨甲蝶呤(methotrexate)可與DHFR緊密結(jié)合使之失去活性，從而阻斷核苷酸生物合成，最終導(dǎo)致細(xì)胞 死亡細(xì)胞培養(yǎng)物經(jīng)氨甲蝶呤處理后，絕大多數(shù)細(xì)胞死亡，但也可篩選到氨甲蝶呤抗性細(xì)胞系吸收氨甲蝶呤能力下降的細(xì)胞系由于DHFR基因拷貝數(shù)的增加，

24、能超量合成DHFR的細(xì)胞系DHFR結(jié)構(gòu)發(fā)生改變，降低了對(duì)氨甲蝶呤親和力的細(xì)胞系細(xì)胞內(nèi)擴(kuò)增DHFR基因時(shí)，與該基因鄰近的染色體DNA片段同樣被擴(kuò)增外源基因在動(dòng)物細(xì)胞內(nèi)的高效表達(dá)3. 新霉素磷酸轉(zhuǎn)移酶基因(neo)neomycin, kanamycin, G4184. 氯霉素乙酰轉(zhuǎn)移酶基因（CAT）真核細(xì)胞不含有內(nèi)源的CAT酶活性將來自于大腸桿菌轉(zhuǎn)座子Tn9的CAT基因接上合適的真核生物啟動(dòng)子和多聚腺苷酸位點(diǎn)CAT酶活性可進(jìn)行精確的定量測定5. Green Fluorescent Protein (GFP)第四節(jié) 轉(zhuǎn)基因動(dòng)物Transgenic AnimalsMammals：Mice, Rats,

25、 Rabbits, Sheep, Goats, Pigs, Cattle etcNon-Mammals：Nematodes線蟲, Insects, Amphibians兩棲動(dòng)物, Fish etc轉(zhuǎn)基因動(dòng)物的用途Current productive livestock- produced through selective breeding- takes generations- incremental increases in desirable traits- high milk yield, high rate of weightgain, desirable wool/hair cha

26、racteristicsUse rDNAtechnology to introduce genes encoding desirable traits into cells of livestock animalsProductivityImproved food quality - manipulation of biosynthetic pathways. Reduced or improved fat composition, overall body composition (lean/fat ratio).Improved food quantity - feed efficiency, rate of gain, overall body composition and size.Other natura