不同轉(zhuǎn)染方法轉(zhuǎn)染牛體細(xì)胞效率的比較

1、農(nóng)業(yè)生物技術(shù)學(xué)報(bào),2011年,第19卷,第6期,第10271033頁Journal of Agricultural Biotechnology,2011,Vol.19,No.6,10271033研究報(bào)告A Letter不同轉(zhuǎn)染方法轉(zhuǎn)染牛體細(xì)胞效率的比較李松丁方榮戴蘊(yùn)平*朱士恩*中國農(nóng)業(yè)大學(xué)動(dòng)物科技學(xué)院,中國農(nóng)業(yè)大學(xué)農(nóng)業(yè)生物技術(shù)國家重點(diǎn)實(shí)驗(yàn)室,北京濟(jì)普霖生物技術(shù)有限公司,北京100193*通訊作者,daiyunping;zhushien摘要為了從新轉(zhuǎn)染方法與傳統(tǒng)轉(zhuǎn)染方法的比較中找出更為高效的轉(zhuǎn)染方法,本研究以綠色熒光蛋白(GFP質(zhì)粒DNA為外源基因,分別采用核轉(zhuǎn)染法、電穿孔法和脂質(zhì)體法轉(zhuǎn)染牛的3

2、種常用核移植供體細(xì)胞(胎兒成纖維細(xì)胞、胎兒輸卵管上皮細(xì)胞、卵丘顆粒細(xì)胞。針對(duì)不同類型的細(xì)胞,實(shí)驗(yàn)首先對(duì)核轉(zhuǎn)染法和電穿孔法的轉(zhuǎn)染參數(shù)進(jìn)行了系統(tǒng)的摸索和優(yōu)化,然后將優(yōu)化后的核轉(zhuǎn)染法、電穿孔法與脂質(zhì)體法在可控實(shí)驗(yàn)條件完全一致的情況下進(jìn)行了轉(zhuǎn)染對(duì)比實(shí)驗(yàn),分析比較了轉(zhuǎn)染48h后的綠色熒光比率和細(xì)胞存活率。結(jié)果顯示,核轉(zhuǎn)染法轉(zhuǎn)染胎兒成纖維細(xì)胞、胎兒輸卵管上皮細(xì)胞和卵丘顆粒細(xì)胞的優(yōu)化程序分別是V013、T023和U023;在電穿孔法中,當(dāng)電場(chǎng)強(qiáng)度為90V/mm,脈沖時(shí)間為10ms時(shí),胎兒成纖維細(xì)胞和卵丘顆粒細(xì)胞的轉(zhuǎn)染效率最高,胎兒輸卵管上皮細(xì)胞則在電場(chǎng)強(qiáng)度為80V/mm,脈沖時(shí)間為5ms時(shí)獲得了最佳轉(zhuǎn)染效率

3、;最后通過轉(zhuǎn)染對(duì)比實(shí)驗(yàn)得出,核轉(zhuǎn)染法在3種細(xì)胞中都獲得了最高的轉(zhuǎn)染效率,分別為(98.78±0.30%、(88.43±2.10%和(71.31±0.77%,均顯著高于電穿孔法和脂質(zhì)體法;轉(zhuǎn)染后的存活率則為脂質(zhì)體法最高,電穿孔法最低。研究結(jié)果說明,核轉(zhuǎn)染法可以成為一種結(jié)合牛體細(xì)胞克隆技術(shù)生產(chǎn)轉(zhuǎn)基因牛更有效的轉(zhuǎn)染方法。關(guān)鍵詞細(xì)胞轉(zhuǎn)染,核轉(zhuǎn)染法,電穿孔法,脂質(zhì)體法,牛體細(xì)胞Comparison of Different Methods for Transfection Efficiency of Bovine Somatic CellsLi Song Ding Fang

4、rong Dai Yunping*Zhu Shien*Animal Sci-tech College,State Key Laboratory for Agrobiotechnology,China Agricultural University,Geneprint Biotechnology Co.LTD,Beijing 100193,China*Corresponding author,daiyunping;zhushienDOI:10.3969/j.issn.1674-7968.2011.06.007Abstact The objective of this study was to f

5、ind a more efficient transfection method between a new transfection method and the traditional transfection methods.Three types of bovine donor cells(fetal fibroblast cells,fetal oviduct epithelial cells and cumulus granulosa cellsthat commonly used for nuclear transfer were transfected with plasmid

6、 DNA of green fluorescent protein(GFPgene using the nucleofection,electroporation and lipofection.We firstly determined optimal transfection parameters for nucleofection and electroporation,then transfection experiments were carried out to compare efficiency of optimized nucleofection and electropor

7、ation with lipofection under identical conditions,and green fluorescence ratio and cell survival were assessed48h after transfection.The results showed that the optimization program of nucleofection for fetal fibroblasts,fetal oviduct epithelial cells and cumulus granulosa cells were V013,T023and U0

8、23;In electroporation,when the electric field was90V/mm,pulse duration was10ms,fetal fibroblasts and cumulus granulosa cells got the highest efficiency, while parameters of80V/mm and5ms were good for fetal oviduct epithelial cells;Finally,after transfection comparision experiments,the nucleofection

9、demonstrated the highest transfection efficiency of(98.78±0.30%、DOI:10.3969/j.issn.1674-7968.2011.06.007基金項(xiàng)目:本研究由國家轉(zhuǎn)基因生物新品種培育重大專項(xiàng)(No.2008ZX08007-001資助收稿日期:2011-05-16接受日期:2011-08-03農(nóng)業(yè)生物技術(shù)學(xué)報(bào)Journal of Agricultural Biotechnology(88.43±2.10%and(71.31±0.77%respectively in all three types o

10、f cells,were significantly higher than electroporation and lipofection;and lipofection yielded the highest survival rate,electroporation got the lowest. The results demonstrate that nucleofection can be a more efficient alternative to produce transgenic cattle when combining with somatic cell clonin

11、g technology.Keywords Cell transfection,Nucleofection,Electroporation,Lipofection,Bovine somatic cells細(xì)胞轉(zhuǎn)染技術(shù)是采用一定的方法和途徑將外源分子如DNA、RNA等導(dǎo)入特定的細(xì)胞,并表達(dá)目的基因,產(chǎn)生特定功能的蛋白質(zhì)分子。在生產(chǎn)轉(zhuǎn)基因動(dòng)物的研究中,通過結(jié)合細(xì)胞轉(zhuǎn)染技術(shù)與體細(xì)胞克隆技術(shù)培育轉(zhuǎn)基因動(dòng)物是一條非常有效的途徑,選擇合適的細(xì)胞轉(zhuǎn)染方法并進(jìn)行優(yōu)化,將會(huì)得到較高的轉(zhuǎn)染效率,從而可以間接提高轉(zhuǎn)基因克隆技術(shù)的效率。目前細(xì)胞轉(zhuǎn)染應(yīng)用最為普遍的是脂質(zhì)體法(Fu et al.,2008;Iguma e

12、t al.,2005;Lee et al.,2005和電穿孔法(Lee et al.,2005;Roh et al.,2000。陽離子脂質(zhì)體作為一種有效的基因傳遞載體具有下列特點(diǎn):對(duì)細(xì)胞類型有選擇性,轉(zhuǎn)染效果隨細(xì)胞類型變化大,對(duì)DNA的質(zhì)量要求高,但操作簡(jiǎn)便,成本適中且細(xì)胞毒性低。電穿孔技術(shù)在20世紀(jì)80年代初期開始用于將DNA導(dǎo)人多種動(dòng)物細(xì)胞,理論上可以轉(zhuǎn)染幾乎所有類型細(xì)胞。較之脂質(zhì)體轉(zhuǎn)染,電穿孔法有使用方便,轉(zhuǎn)染效率高,外源基因通常以單拷貝形式整合(Boggs et al.,1986等諸多優(yōu)點(diǎn),但伴隨較高的細(xì)胞毒性。近些年由Amaxa公司研發(fā)出的核轉(zhuǎn)染技術(shù)是一種非病毒介導(dǎo)的新型轉(zhuǎn)染技術(shù),該

13、技術(shù)在傳統(tǒng)電穿孔技術(shù)的基礎(chǔ)上進(jìn)行改良,設(shè)計(jì)出包含針對(duì)不同類型細(xì)胞的優(yōu)化轉(zhuǎn)染程序的Amaxa R Nucleofecto R 轉(zhuǎn)染設(shè)備和細(xì)胞特異性核轉(zhuǎn)染試劑。核轉(zhuǎn)染法就是采用對(duì)特定類型細(xì)胞配套的核轉(zhuǎn)染試劑和轉(zhuǎn)染程序,直接把外源基因?qū)爰?xì)胞核中,以達(dá)到最佳轉(zhuǎn)染效率。與其他轉(zhuǎn)染技術(shù)相比,其可以直接將DNA轉(zhuǎn)染到細(xì)胞核內(nèi),實(shí)現(xiàn)了因受到細(xì)胞分裂限制而一直困擾人們的原代細(xì)胞的高效率轉(zhuǎn)染,所以特別適用于轉(zhuǎn)染原代細(xì)胞和難以轉(zhuǎn)染的細(xì)胞系(Cao et al., 2009;Ganesh et al.,2003;Gresch et al.,2004;Leclere et al.,2005;Lenz et al.,2

14、003;Trompeter et al.,2003; Yin et al.,2006。目前該技術(shù)較多的應(yīng)用于腫瘤研究、免疫學(xué)、組織工程學(xué)及心血管疾病研究領(lǐng)域中,還未見有用于牛體細(xì)胞轉(zhuǎn)染的相關(guān)報(bào)道。胎兒成纖維細(xì)胞、胎兒輸卵管上皮細(xì)胞和卵丘顆粒細(xì)胞都是常用的核移植供體細(xì)胞,并且均已成功獲得克隆牛(Cibelli et al.,1998;Kato et al.,2000;Zakhartchenko et al.,1999,本實(shí)驗(yàn)應(yīng)用帶有綠色熒光蛋白基因的質(zhì)粒在上述三種細(xì)胞上進(jìn)行轉(zhuǎn)染,更系統(tǒng)地比較這三種轉(zhuǎn)染方法的轉(zhuǎn)染效率,為進(jìn)一步的穩(wěn)定轉(zhuǎn)染、篩選轉(zhuǎn)基因細(xì)胞并生產(chǎn)轉(zhuǎn)基因克隆牛提供基礎(chǔ)資料。1結(jié)果與分析1

15、.1牛的各種體細(xì)胞系的建立經(jīng)過原代培養(yǎng)、繼代培養(yǎng)、冷凍保存等體外培養(yǎng)操作,成功的建立了黃牛胎兒成纖維細(xì)胞系(編號(hào)LXH102FFb、奶牛胎兒輸卵管上皮細(xì)胞系(編號(hào)1001FOV和黃牛卵丘顆粒細(xì)胞系(編號(hào)CC(圖1。1.2核轉(zhuǎn)染法轉(zhuǎn)染條件的優(yōu)化對(duì)LXH102FFb、1001FOV和CC的推薦核轉(zhuǎn)染程序進(jìn)行篩選。LXH102FFb和CC都采用程序A024、T016、U012、U023和V013轉(zhuǎn)染,1001FOV采用程序S005、T013、T020、T023和U017轉(zhuǎn)染,從圖2可以看出,各細(xì)胞在推薦程序下轉(zhuǎn)染后的存活率差異不大,以轉(zhuǎn)染后48h綠色熒光細(xì)胞百分比高低為標(biāo)準(zhǔn)進(jìn)行選擇得出,LXH102

16、FFb、1001FOV和CC的最優(yōu)轉(zhuǎn)染程序分別是V013、T023和U023。LXH102FFb1001FOVCC圖1牛3種體細(xì)胞系Figure1Three types of bovine somatic cells1028不同轉(zhuǎn)染方法轉(zhuǎn)染牛體細(xì)胞效率的比較Comparison of Different Methods for Transfection Efficiency of Bovine Somatic Cells圖3不同電轉(zhuǎn)參數(shù)下牛三種體細(xì)胞的轉(zhuǎn)染效率和存活率Figure 3Transfection efficiency and survival rate ofLXH102FFb (A

17、,1001FOV (Band CC (Cunder different electroporation parametersA.1:140V,5ms;2:140V,8ms;3:140V,10ms;4:160V,5ms;5:160V,8ms;6:160V,10ms;7:180V,5ms;8:180V,8ms;9:180V,10msB.1:120V,1ms;2:120V,2ms;3:120V,5ms;4:140V,1ms;5:140V,2ms;6:140V,5ms;7:160V,1ms;8:160V,2ms;9:160V,5msC.1:140V,5ms;2:140V,8ms;3:140V,10ms

18、;4:160V,5ms;5:160V,8ms;6:160V,10ms;7:180V,5ms;8:180V,8ms;9:180V,10ms圖2不同核轉(zhuǎn)染程序下牛三種體細(xì)胞的轉(zhuǎn)染效率和存活率Figure 2Transfection efficiency and survival rate of LXH102FFb (A,1001FOV (Band CC (Cunder different nucleofection procedures1.3電穿孔法轉(zhuǎn)染條件的優(yōu)化通過調(diào)整電脈沖時(shí)間(110ms和改變電場(chǎng)強(qiáng)度(6090V/mm來設(shè)置電轉(zhuǎn)參數(shù)組合轉(zhuǎn)染LXH102FFb 、1001FOV 和CC ,不同

19、參數(shù)組合下各細(xì)胞的轉(zhuǎn)染效率見圖3,當(dāng)電壓為180V ,脈沖時(shí)間為10ms 時(shí),LXH102FFb 和CC 的轉(zhuǎn)染效率最高,1001FOV 則在電壓為160V ,脈沖時(shí)間為5ms 時(shí)獲得了最高轉(zhuǎn)染效率,為了更好的進(jìn)行后面的轉(zhuǎn)染對(duì)比實(shí)驗(yàn),選擇轉(zhuǎn)染效率最高的參數(shù)組合為最優(yōu)參數(shù),盡管這些細(xì)胞在獲得最高轉(zhuǎn)染效率的同時(shí)存活率普遍偏低。1.4不同轉(zhuǎn)染方法轉(zhuǎn)染效率的比較如圖4所示,在LXH102FFb 和1001FOV 的轉(zhuǎn)染對(duì)比實(shí)驗(yàn)中,3種轉(zhuǎn)染方法產(chǎn)生的轉(zhuǎn)染效率及細(xì)胞存活率兩兩之間均差異顯著(LXH102FFb,P <0.05;1001FOV,P <0.01,核轉(zhuǎn)染法在3種細(xì)胞中都獲得了最高的轉(zhuǎn)

20、染效率,分別為(98.78±0.30%、(88.43±2.10%和(71.31±0.77%,均顯著高于電穿孔法的(75.14±2.95%、(21.16±2.44%、(36.62±6.20%和脂質(zhì)體法的(65.16±1.53%、(2.57±0.26%、(18.25±0.33%;另外,轉(zhuǎn)染后各細(xì)胞的存活率則為脂質(zhì)體法最高,并且與其它2種方法得到的存活率差異顯著(P <0.05或P <0.01。2討論隨著科學(xué)技術(shù)的不斷進(jìn)步,將外源基因?qū)胝婧思?xì)胞的轉(zhuǎn)染技術(shù)在生物醫(yī)藥研究和基因功能研究轉(zhuǎn)染效率Tran

21、sfection efficiency 100806040200A024T016U012U023V013核傳染程序Nucleofection programsA 百分比/%P e r c e n t a g e存活率Survival rate核傳染程序Nucleofection programsS005100806040200T013T020T023U017B百分比/%P e r c e n t a g e轉(zhuǎn)染效率Transfection efficiency 存活率Survival rate100806040200A024核傳染程序Nucleofection programsT016U012

22、U023V013C百分比/%P e r c e n t a g e轉(zhuǎn)染效率Transfection efficiency 存活率Survival rateA百分比/%P e r c e n t a g e100806040200電轉(zhuǎn)參數(shù)組合體(電壓,脈沖時(shí)間Parameters for electroporation(voltage,pulse time轉(zhuǎn)染效率Transfection efficiency 存活率Survival rate123456789B百分比/%P e r c e n t a g e100電轉(zhuǎn)參數(shù)組合體(電壓,脈沖時(shí)間Parameters for electropora

23、tion(voltage,pulse time806040200轉(zhuǎn)染效率Transfection efficiency 存活率Survival rate123456789C百分比/%P e r c e n t a g e100電轉(zhuǎn)參數(shù)組合體(電壓,脈沖時(shí)間Parameters for electroporation(voltage,pulse time806040200轉(zhuǎn)染效率Transfection efficiency 存活率Survival rate1234567891029農(nóng)業(yè)生物技術(shù)學(xué)報(bào)Journal of Agricultural Biotechnology方面起著越來越重要的作用

24、,選擇好的轉(zhuǎn)染方法,對(duì)條件進(jìn)行優(yōu)化,將會(huì)得到較高的轉(zhuǎn)染效率。化學(xué)轉(zhuǎn)染方法經(jīng)過多年的發(fā)展已經(jīng)得到很好的提高,而物理轉(zhuǎn)染方法的改良進(jìn)展卻很有限(Colosimo et al.,2000。Amaxa 公司在傳統(tǒng)電穿孔技術(shù)的基礎(chǔ)上加以改良形成了一種新的物理轉(zhuǎn)染方法-核轉(zhuǎn)染,依靠先進(jìn)的電穿孔設(shè)備設(shè)定特定轉(zhuǎn)染程序并結(jié)合使用配套的特異性轉(zhuǎn)染液直接把外源基因?qū)爰?xì)胞核,轉(zhuǎn)染效率不受細(xì)胞分裂快慢影響,與以往的物理方法相比該方法的轉(zhuǎn)染效果有了質(zhì)的飛躍。從核轉(zhuǎn)染儀公司的官方網(wǎng)站(的最優(yōu)轉(zhuǎn)染程序,但是本實(shí)驗(yàn)中的細(xì)胞系并未羅列在特定的細(xì)胞系范圍內(nèi),而是作為泛指細(xì)胞類型被網(wǎng)站建議對(duì)多個(gè)推薦程序進(jìn)行篩選。Maurisse

25、等(2010經(jīng)過篩選多種轉(zhuǎn)染程序發(fā)現(xiàn)豬胎兒成纖維細(xì)胞最優(yōu)轉(zhuǎn)染程序?yàn)閁020,獲得的轉(zhuǎn)染效率是90%,兔胎兒成纖維細(xì)胞最優(yōu)轉(zhuǎn)染程序?yàn)閁023,轉(zhuǎn)染效率為38%;也有人轉(zhuǎn)染豬胎兒成纖維細(xì)胞時(shí)對(duì)推薦的10個(gè)轉(zhuǎn)染程序進(jìn)行篩選得出最優(yōu)程序U023,獲得了79%的轉(zhuǎn)染效率(Nakayama et al.,2007。在本實(shí)驗(yàn)中,首次用核轉(zhuǎn)染法轉(zhuǎn)染牛體細(xì)胞并篩選了轉(zhuǎn)染程序,LXH102FFb 和CC 均采用成纖維細(xì)胞推薦程序A024、T016、U012、U023和V013進(jìn)行轉(zhuǎn)染比較和篩選,1001FOV 則從上皮細(xì)胞推薦程序S005、T013、T020、T023和U017中篩選,在優(yōu)化過程中發(fā)現(xiàn)轉(zhuǎn)染后的存活

26、率差異不大,于是選擇轉(zhuǎn)染后48h 綠色熒光細(xì)胞比例最高的為最優(yōu)轉(zhuǎn)染程序。從圖2得出LXH102FFb 的最優(yōu)轉(zhuǎn)染程序是V013,轉(zhuǎn)染效率為(96.91±1.88%,明顯高于上述豬和兔成纖維細(xì)胞的轉(zhuǎn)染效率,可見,不同細(xì)胞個(gè)體的優(yōu)化程序和轉(zhuǎn)染效率有很大的差異。而1001FOV 和CC 的最優(yōu)程序分別為T023(82.92±0.31%和U023(72.70±2.57%。電穿孔法可廣泛應(yīng)用于各類細(xì)胞的轉(zhuǎn)染(Shigekawa and Dower,1988,但好的電轉(zhuǎn)參數(shù)因細(xì)胞不同而異。為達(dá)到較好的轉(zhuǎn)染效果,一般考慮對(duì)電場(chǎng)強(qiáng)度和脈沖時(shí)間這兩個(gè)關(guān)鍵因素進(jìn)行優(yōu)化(Rols an

27、d Teissie,1998,在眾多關(guān)于牛體細(xì)胞克隆文獻(xiàn)中(Arat et al.,2001;Forsberg et al.,2002;Gong et al.,2004;Roh et al.,2000都有報(bào)道使用電穿孔法轉(zhuǎn)染,但并沒有提及對(duì)電轉(zhuǎn)參數(shù)的優(yōu)化和對(duì)轉(zhuǎn)染效率高低的評(píng)估。Jordan 等(2008對(duì)指數(shù)型電穿孔儀轉(zhuǎn)染各種難轉(zhuǎn)的人細(xì)胞的電擊參數(shù)進(jìn)行了系統(tǒng)的摸索和探討,在通過轉(zhuǎn)染siRNA 進(jìn)行評(píng)估的過程中,人原代成纖維細(xì)胞和人靜脈內(nèi)皮細(xì)胞的轉(zhuǎn)染效率最高達(dá)到93%和94%,當(dāng)對(duì)人原代成纖維細(xì)胞轉(zhuǎn)染GFP 質(zhì)粒時(shí)則獲得了44%的最好轉(zhuǎn)染效率。Ross 等(2009在豬胎兒成纖維細(xì)胞上對(duì)方波形式

28、的電穿孔方法進(jìn)行了優(yōu)化,發(fā)現(xiàn)細(xì)胞系和電壓對(duì)轉(zhuǎn)染效率影響顯著,同時(shí)電壓還顯著的影響細(xì)胞存活率。優(yōu)化過程中電壓以50V 的增幅從100V 提高到350V ,細(xì)胞的GFP 陽性率從(3.2±0.8%升高到(43.0±3.4%,而存活率卻從(90.5±8.0%降低到(44.8±2.0%。本實(shí)驗(yàn)根據(jù)以往經(jīng)驗(yàn)和BTX 代理商推薦的電擊參數(shù),分別用6090V/mm 和l10ms 的參數(shù)組合轉(zhuǎn)染牛體細(xì)胞,并系統(tǒng)的比較了各細(xì)胞的轉(zhuǎn)染效率和存活率,實(shí)驗(yàn)發(fā)現(xiàn)各圖4不同轉(zhuǎn)染方法牛三種體細(xì)胞的轉(zhuǎn)染效率和存活率同類圖例上字母不同(a ,b ,c 或A ,B ,C表示差異顯著(P &

29、lt;0.05或極顯著(P <0.01Figure 4Transfection efficiency and survival rate of LXH102FFb (A,1001FOV (Band CC (Cunder different transfection methodsValues with different superscripts in the same type of column indicate significant difference or extremely significant difference (a vs b vs c,P <0.05or A

30、 vs B vs C,P <0.01百分比/%P e r c e n t a g eA100806040200核轉(zhuǎn)染Nucleofection (V013電穿孔Electroporation (180V,10ms脂質(zhì)體Lipofection轉(zhuǎn)染效率Transfection efficiency 存活率Survival rateaab cbc B百分比/%P e r c e n t a g e80核轉(zhuǎn)染Nucleofection (T023脂質(zhì)體Lipofection1006040200轉(zhuǎn)染效率Transfection efficiency存活率Survival rateAABBC C 電

31、穿孔Electroporation (160V,5msC百分比/%P e r c e n t a g e801006040200轉(zhuǎn)染效率Transfection efficiency存活率Survival rateAABACC 核轉(zhuǎn)染Nucleofection (U023電穿孔Electroporation (180V,10ms脂質(zhì)體Lipofection1030細(xì)胞的轉(zhuǎn)染效率和存活率變化很廣,各細(xì)胞轉(zhuǎn)染效率隨著電壓和脈沖時(shí)間的升高而增長(zhǎng),最低的是(0.41±0.08%,最高的為(75.03±2.31%;存活率則急劇下降,范圍為(90.12±4.47%(23.70

32、±2.47%選擇轉(zhuǎn)染效率最高的參數(shù)組合為最優(yōu)參數(shù),LXH102FFb 和CC的最優(yōu)參數(shù)為90V/mm,10ms,1001FOV的為80V/mm,5ms。采用本研究所描述的3種轉(zhuǎn)染方法轉(zhuǎn)染牛體細(xì)胞的對(duì)比實(shí)驗(yàn)?zāi)壳斑€未見相關(guān)報(bào)道,但在其它類型細(xì)胞中類似的研究實(shí)驗(yàn)已經(jīng)有很多,Hamm等(2002、Jacobsen等(2006和Maurisse等(2010比較核轉(zhuǎn)染法與脂質(zhì)體法轉(zhuǎn)染人和其它哺乳動(dòng)物多種細(xì)胞的轉(zhuǎn)染效果,結(jié)果核轉(zhuǎn)染法獲得最高的轉(zhuǎn)染效率; Nakayama等(2007在豬胎兒成纖維細(xì)胞上比較核轉(zhuǎn)染法、電穿孔法和脂質(zhì)體法的轉(zhuǎn)染效率,各方法優(yōu)化后分別獲得了79.0、53.4和8.2%的最

33、佳轉(zhuǎn)染效率,核轉(zhuǎn)染法的效率明顯最高,與本實(shí)驗(yàn)結(jié)果一致,而且作者將核轉(zhuǎn)染后篩選得到的轉(zhuǎn)基因細(xì)胞用于體細(xì)胞克隆;在人胚胎干細(xì)胞的轉(zhuǎn)染研究中;Cao等(2009在上述3種方法的基礎(chǔ)上再加入了病毒轉(zhuǎn)染法進(jìn)行轉(zhuǎn)染對(duì)比實(shí)驗(yàn),雖然核轉(zhuǎn)染法并沒有獲得最高的轉(zhuǎn)染效率,依然被作者推薦。本實(shí)驗(yàn)在LXH102FFb、1001FOV和CC這3種細(xì)胞中分別采用核轉(zhuǎn)染程序V013、T023和U023獲得了最高的轉(zhuǎn)染效率(98.78±0.30%、(88.43±2.10%和(71.31±0.77%,均顯著高于電穿孔法和脂質(zhì)體法,轉(zhuǎn)染后存活率的比較結(jié)果為脂質(zhì)體法>核轉(zhuǎn)染法>電穿孔法。綜合

34、考慮轉(zhuǎn)染效果,我們認(rèn)為核轉(zhuǎn)染是值得推薦的高效轉(zhuǎn)染方法?？傊?核轉(zhuǎn)染法作為一種非病毒的高效轉(zhuǎn)染方法應(yīng)用于牛體細(xì)胞轉(zhuǎn)染是切實(shí)可行的,為下一步的穩(wěn)定轉(zhuǎn)染和篩選轉(zhuǎn)基因細(xì)胞打下基礎(chǔ),相信該轉(zhuǎn)染方法終將用于轉(zhuǎn)基因克隆牛的生產(chǎn)并很可能提高轉(zhuǎn)基因克隆的整體效率。3材料與方法3.1化學(xué)試劑與實(shí)驗(yàn)材料pmaxGFP質(zhì)粒和核轉(zhuǎn)染試劑為L(zhǎng)onza公司(德國產(chǎn)品;脂質(zhì)體(Lipofectamine TM2000為Invitrogen 公司(美國產(chǎn)品;胎牛血清(FBS、胰蛋白酶(Trypsin、DMEM和PBS購自Gibco公司(美國;細(xì)胞凍存液(cellBanker2購自ZENOAQ公司(日本;其他藥品除特殊注明外均購

35、自Sigma公司(美國。細(xì)胞培養(yǎng)器皿購自Corning或Nunc公司(美國。Holstein 奶牛胎兒取自山東省農(nóng)業(yè)科學(xué)院奶牛研究中心,黃牛胎兒取自山東科龍畜牧產(chǎn)業(yè)有限公司的牛場(chǎng),黃牛卵巢來自北京周邊地區(qū)屠宰場(chǎng)。3.2牛的各種體細(xì)胞系的建立分別取1頭3月齡Holstein奶牛胎兒和1頭2月齡黃牛胎兒建立奶牛胎兒輸卵管上皮細(xì)胞系(1001FOV和黃牛胎兒成纖維細(xì)胞系(LXH102FFb,組織樣被剪碎成1mm3左右的小塊,PBS清洗2次后分批植塊于含6mL DMEM+20%FBS的25cm2的培養(yǎng)瓶中。以黃牛卵巢組織樣建立黃牛卵丘顆粒細(xì)胞系(CC,將清理過的卵巢置于培養(yǎng)皿中,用10 mL一次性注射

36、器抽吸卵巢表面26mm大小卵泡中的卵泡液,回收形態(tài)均勻、結(jié)構(gòu)致密的卵丘-卵母細(xì)胞-復(fù)合體(COCs,PBS清洗2遍后以0.1%透明質(zhì)酸酶溶液震蕩3min,DMEM+10%FBS洗滌離心后重懸至1×105個(gè)/mL,6mL細(xì)胞溶液鋪至25cm2的培養(yǎng)瓶中。牛胎兒組織樣和卵丘顆粒細(xì)胞于37.5,5%CO2培養(yǎng)箱培養(yǎng)67d,每2d換液1次,待細(xì)胞生長(zhǎng)匯合后,以0.1%胰蛋白酶消化傳代23次,分批凍存,先放置負(fù)80度冰箱過夜,再投入液氮長(zhǎng)期保存。3.3核轉(zhuǎn)染法轉(zhuǎn)染將冷凍保存的細(xì)胞解凍后傳代2次,培養(yǎng)至70%80%匯合,0.1%胰蛋白酶消化收集細(xì)胞并計(jì)數(shù),用核轉(zhuǎn)染液重懸細(xì)胞(細(xì)胞特異性核轉(zhuǎn)染試劑

37、Basic Nucleofector Solution和Supplement混合得到核轉(zhuǎn)染液,兩者體積比為4.51,加入pmaxGFP質(zhì)粒混勻,把混合液轉(zhuǎn)移到電擊杯。按照核轉(zhuǎn)染儀Amaxa R Nucleofector R的操作手冊(cè)選擇與各類細(xì)胞對(duì)應(yīng)的推薦轉(zhuǎn)染程序進(jìn)行轉(zhuǎn)染,完成轉(zhuǎn)染后,向電擊杯中加入200L預(yù)熱的培養(yǎng)液,輕輕吸打,用吸管將細(xì)胞混合液轉(zhuǎn)移到含5mL預(yù)熱培養(yǎng)液的培養(yǎng)皿中放入CO2培養(yǎng)箱培養(yǎng)。48h后參照陰性對(duì)照分析轉(zhuǎn)染效率和細(xì)胞存活率。每次核轉(zhuǎn)染的細(xì)胞數(shù)量設(shè)為78×105個(gè),核轉(zhuǎn)染液體積為100L,pmaxGFP 質(zhì)粒2g。3.4電穿孔法轉(zhuǎn)染將冷凍保存的細(xì)胞解凍后傳代2次

38、,培養(yǎng)至70%80%匯合,0.1%胰蛋白酶消化收集細(xì)胞,并進(jìn)行細(xì)胞計(jì)數(shù)。加入電轉(zhuǎn)緩沖液(PBS中含272mmol/L蔗糖、7mmol/L K2HPO4,1mmol/L MgCl2,pH7.4重懸不同轉(zhuǎn)染方法轉(zhuǎn)染牛體細(xì)胞效率的比較Comparison of Different Methods for Transfection Efficiency of Bovine Somatic Cells10311032 Journal of Agricultural Biotechnology 率， 確定與之對(duì)應(yīng)的最佳轉(zhuǎn)染程序。 實(shí)驗(yàn) 2： BTX 電穿孔儀(ECM2001設(shè)置不同 用 加入 2 mm 電

39、擊杯中， 將電擊杯放入電擊槽， BTX 參數(shù)組合對(duì)牛的 3 種體細(xì)胞進(jìn)行電穿孔轉(zhuǎn)染實(shí)驗(yàn)， 用 電穿孔儀(ECM2001設(shè)置相應(yīng)的參數(shù)組合進(jìn)行電擊， 比較各細(xì)胞在不同參數(shù)組合下的轉(zhuǎn)染效率，找到與 電擊后室溫靜置 2 min， 向電擊杯中加入 200 L 預(yù)熱 之對(duì)應(yīng)的最佳轉(zhuǎn)染參數(shù)。 實(shí)驗(yàn) 3： 針對(duì)牛的 3 種體細(xì)胞， 將優(yōu)化后的核轉(zhuǎn) 的培養(yǎng)液， 輕輕吸打， 將細(xì)胞轉(zhuǎn)移到含 5 mL 預(yù)熱培養(yǎng) 48 液的培養(yǎng)皿中，置 CO2 培養(yǎng)箱培養(yǎng)， h 后參照陰性 染法、電穿孔法與脂質(zhì)體法在可控實(shí)驗(yàn)條件完全一 致的情況下進(jìn)行轉(zhuǎn)染對(duì)比實(shí)驗(yàn)，比較不同轉(zhuǎn)染方法 對(duì)照分析轉(zhuǎn)染效率和細(xì)胞存活率。 的轉(zhuǎn)染效率和毒性。

40、 3.5 脂質(zhì)體法轉(zhuǎn)染 統(tǒng)計(jì)學(xué)分析：所有實(shí)驗(yàn)至少重復(fù) 3 次，采用 TM 參照產(chǎn)品 Lipofectamine 2000 使用說明書， 在 SPSS19.0 統(tǒng)計(jì)學(xué)軟 件對(duì)數(shù)據(jù)進(jìn) 行統(tǒng)計(jì)學(xué) 分析， 計(jì) 調(diào)整細(xì)胞密度至 5× 量資料所有數(shù)據(jù)均以平均數(shù)(x±標(biāo)準(zhǔn)差(SD表示。 轉(zhuǎn)染前一天, 6 孔板中接種細(xì)胞， 105/ 孔。 轉(zhuǎn)染的當(dāng)天， 細(xì)胞密度最好達(dá)到 7080， 不同轉(zhuǎn)染方法轉(zhuǎn)染效率的比較采用 ANOVA 檢驗(yàn)， 吸出各孔內(nèi)的培養(yǎng)基， 用無血清 DMEM 培養(yǎng)基漂洗 當(dāng) P0.05 時(shí)認(rèn)為差異顯著， P0.01 時(shí)認(rèn)為差異 1 次， 向各孔內(nèi)加入 Opti-MEM 培養(yǎng)

41、基 2 mL。 在新的 極顯著。 1.5 mL EP 管中準(zhǔn)備溶液 A 和溶液 B， 孔板每孔用 6 量如下， 液： A 250 L Opti-MEM 培 養(yǎng) 基 +2 g 參考文獻(xiàn) B 250 pmaxGFP 質(zhì) 粒 ， 室 溫 下 靜 置 5 min， 液 ： L Arat S., Rzucidlo S.J ., G ibbons J., Miyoshi K., and Stice S.L., Opti-MEM 培 養(yǎng) 基 +10 L 脂 質(zhì) 體 2001, Production of transgenic bovine embryos by transfer LipofectamineT

42、M2000， 室溫下靜置 5 min。將 A， 兩 B of transfected granulosa cells into enucleated oocytes, 種液體混合，室溫下靜置 20 min 后加入到 6 孔板 Molecular Reproduction and Development, 60(1: 2026 中， 輕輕搖晃使之與細(xì)胞均勻接觸， 放入 CO2 培養(yǎng)箱 Boggs S., Gregg R.G., Borenstein N. and Smithies O., 1986, Efficient transformation and frequent single-sit

43、e, single-copy 培養(yǎng)。讓脂質(zhì)體 -DNA 復(fù)合物與細(xì)胞接觸 6 h 后， 換 insertion of DNA can be obtained in mouse erythroleukemia 成含有血清的完全培養(yǎng)基繼續(xù)培養(yǎng)。考慮到轉(zhuǎn)染的 cells transformed by electroporation, Experimental 細(xì)胞密度較大， 隔天進(jìn)行傳代， h 后參照陰性對(duì)照 48 Hematology, 14(10: 988994 分析轉(zhuǎn)染效率和細(xì)胞存活率。 細(xì)胞， 每次電轉(zhuǎn) 78×105 個(gè)細(xì)胞 / 組， 加入 pmaxGFP 質(zhì)粒 2 g，將細(xì)胞懸液

44、及質(zhì)粒混合均勻后取 200 L Cao F., Xie X.Y, Gollan T., Zhao L., Narsinh K., Lee R.J., and human embryonic stem cells, Molecular Imaging and Biology, 12(1: 1524 Cibelli J.B., Stice S.L., Golueke P.J., Kane J.J., Jerry J., Blackwell C., Ponce de León F.A., and Robl J.M., 1998, Cloned transgenic calves produ

45、ced from nonquiescent fetal fibroblasts, Science, 280(5367: 12561258 Colosimo A., Goncz K.K., Holmes A.R., Kunzelmann K., Novelli G., Malone R.W., Bennett M.J., and Gruenert D.C., 2000, Transfer and expression of foreign genes in mammalian 農(nóng)業(yè)生物技術(shù)學(xué)報(bào) 3.6 細(xì)胞存活率分析 每次轉(zhuǎn)染實(shí)驗(yàn)中均設(shè)置未轉(zhuǎn)染的陰性細(xì)胞為對(duì) 照， 而且轉(zhuǎn)染時(shí)各組的細(xì)胞數(shù)量要相同

46、。 轉(zhuǎn)染后培養(yǎng) 48 h， 消化各組細(xì)胞準(zhǔn)備流式分析時(shí)分別進(jìn)行細(xì)胞計(jì) 數(shù)，然后將各轉(zhuǎn)染組的細(xì)胞數(shù)量與陰性對(duì)照的細(xì)胞 數(shù)量相除即得出轉(zhuǎn)染后 48 h 的細(xì)胞存活率。 3.7 細(xì)胞轉(zhuǎn)染效率分析 轉(zhuǎn)染的細(xì)胞培養(yǎng) 48 h 后，用 PBS 漂洗 2 遍， 再 Wu J.C., 2009, Comparison of gene-transfer efficiency in cells, Biotechniques, 29(2: 3148, 3202, 324 passim 用 0.1胰蛋白酶消化收集細(xì)胞，然后通過流式細(xì)胞 Forsberg E.J., Strelchenko N.S., Augenste

47、in M.L., Betthauser J. 儀 CYFLOW space(德國 PARTEC 公司檢測(cè)綠色熒 M., Childs L.A., Eilertsen K.J., Enos J.M., Forsythe T.M., 光細(xì)胞比例來分析不同轉(zhuǎn)染方法對(duì)各類細(xì)胞的轉(zhuǎn)染 Golueke P.J., Koppang R. W., Lange G., Lesmeister T.L., 效率。使用 FloMax 軟件對(duì)資料進(jìn)行分析， 分析時(shí)根 Mallon K.S., Mell G.D., Misica P.M., Pace M.M., 據(jù)未轉(zhuǎn)染的陰性細(xì)胞的自發(fā)熒光水平設(shè)定門控標(biāo)準(zhǔn)。 Pfist

48、er-Genskow M., Voelker G.R., Watt S.R., and Bishop 3.8 實(shí)驗(yàn)設(shè)計(jì)及數(shù)據(jù)統(tǒng)計(jì) 實(shí)驗(yàn) 1： 使用推薦程序?qū)ε５?3 種體細(xì)胞進(jìn)行核 轉(zhuǎn)染實(shí)驗(yàn)，比較各細(xì)胞在不同轉(zhuǎn)染程序下的轉(zhuǎn)染效 M.D., 2002, Production of cloned cattle from in vitro systems, Biology of Reproduction, 67(1: 327333 Fu J., Guan P., Zhao L., Li H., Huang S., Zeng F., and Zeng Y., 2008, Effects of dono

49、r cells on in vitro development of 不同轉(zhuǎn)染方法轉(zhuǎn)染牛體細(xì)胞效率的比較 1033 Comparison of Different Methods for Transfection Efficiency of Bovine Somatic Cells cloned bovine embryos, Journal of Genetics and Genomics, 35(5: 273278 Ganesh L., Burstein E., Guha-Niyogi A., Louder M.K., Mascola J.R., Klomp L.W.J., Wijmeng

50、a C., Duckett C.S., and Nabel G.J., 2003, The gene product Murr1 restricts HIV-1 replication in resting CD4 lymphocytes, Nature, 426 (6968: 853857 Gong G.C., Dai Y.P., Fan B.L., Zhu H.B., Wang H.P., Wang L.L. , Fang C.G., Wan R., Liu Y., Li R., and Li N., 2004, Production of transgenic blastocyst by

51、 nuclear transfer from different types of somatic cells in cattle, Science in China Series C: Life Sciences, 47(2: 183189 Gresch O., Engel F.B., Nesic D., Tran T.T., England H.M., Hickman E.S., Korner I., Gan L., Chen S., Castro-Obregon S., Hammermann R., Wolf J., Müller-Hartmann H., Nix M., Si

52、ebenkotten G., Kraus G., and Lun K., 2004, New non-viral method for gene transfer into primary cells, Methods, 33(2: 151163 Hamm A., Krott N., Breibach I., Blindt R., and Bosserhoff A.K., 2002, Efficient transfection method for primary cells, Tissue Engineering, 8(2: 235245 Iguma L.T., Lisauskas S.F

53、., Melo E.O., Franco M.M., Pivato I., Vianna G.R., Sousa R.V., Dode M.A., Aragao F.J., Rech E. L., and Rumpf R., 2005, Development of bovine embryos reconstructed by nuclear transfer of transfected and non-transfected adult fibroblast cells, Genetics and Molecular Research, 4(1: 5566 Jacobsen F., Me

54、rtens-Rill J., Beller J., Hirsch T., Daigeler A., Langer S., Lehnhardt M., Steinau H.U., and Steinstraesser L., 2006, Nucleofection: A New method for cutaneous gene Transfer, Journal of Biomedicine and Biotechnology, 2006 (5: 26060 Jordan E.T., Collins M., Terefe J., Ugozzoli L., and Rubio T., 2008,

55、 Optimizing electroporation conditions in primary and other difficult-to-transfect cells, Journal of Biomolecular Techniques: JBT, 19(5: 328334 Kato Y., Tani T., and Tsunoda Y., 2000, Cloning of calves from various somatic cell types of male and female adult, newborn and fetal cows, Reproduction, 12

56、0(2: 231237 Leclere P.G., Panjwani A., Docherty R., Berry M., Pizzey J., and Tonge D.A., 2005, Effective gene delivery to adult neurons by a modified form of electroporation, Journal of Neuroscience Methods, 142(1: 137143 Lee S.L., Ock S.A., Yoo J.G., Kumar B.M., Choe S.Y., and Rho G.J., 2005, Effic

57、iency of gene transfection into donor cells + for nuclear transfer of bovine embryos, Molecular Reproduction and Development, 72(2: 191200 Lenz P., Bacot S.M., Frazier-Jessen M.R., and Feldman G.M., 2003, Nucleoporation of dendritic cells: Efficient gene transfer by electroporation into human monocyte-derived dendritic cells, FEBS Letters, 538(13: 149154 Maurisse R., De Semir D., Emamekhoo H., Bedayat B., Abdolmohammadi A., Parsi H., and Gruenert D.C., 2