本文選題：慢病毒載體法 + 轉(zhuǎn)基因小鼠��； 參考：《南方醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 遺傳工程小鼠的功用小鼠因其一些獨(dú)特特點(diǎn)和優(yōu)勢(shì)已成為研究基因功能、開(kāi)展發(fā)育生物學(xué)研究、建造人類(lèi)疾病動(dòng)物模型及研究人類(lèi)疾病發(fā)病機(jī)制等的最重要的和最佳的模式實(shí)驗(yàn)動(dòng)物。識(shí)別基因功能最有效方法是觀察基因功能缺失或基因過(guò)表達(dá)后,細(xì)胞和機(jī)體所產(chǎn)生的表型變化。利用轉(zhuǎn)基因(Transgene)和基因打靶(Gene targeting)[包括基因敲除(Gene knockout)和基因敲入(Geneknockin)等]等技術(shù)產(chǎn)生的遺傳工程小鼠(Genetically modified mice,GMMs)是最具研究?jī)r(jià)值的工具之一。GMMs已成為在整體動(dòng)物水平研究人體發(fā)育、解析基因功能或疾病相關(guān)基因功能,研究人類(lèi)疾病發(fā)病機(jī)制,解答特定人群對(duì)某種疾病的易感性,識(shí)別藥靶,新藥篩選和療效判定等最為有效的手段。 慢病毒載體法制備轉(zhuǎn)基因小鼠的優(yōu)勢(shì)原核顯微注射法為目前最經(jīng)典的和最成熟制備轉(zhuǎn)基因小鼠的方法,被廣泛采用,但是顯微注射制備轉(zhuǎn)基因動(dòng)物的效率很低。近年來(lái),隨著不同用途慢病毒載體的問(wèn)世,慢病毒介導(dǎo)的轉(zhuǎn)基因動(dòng)物制作方法逐漸受到人們青睞;迄今,利用慢病毒載體法已成功制備了轉(zhuǎn)基因小鼠、大鼠、豬、牛和雞等,效率較以往的方法高許多,該方法已被證明是普遍適用于從哺乳類(lèi)動(dòng)物到禽類(lèi)的轉(zhuǎn)基因動(dòng)物制作方法。與原核顯微注射法相比,慢病毒載體法有如下優(yōu)勢(shì):(1)原核顯微注射時(shí)注射針必須插入核膜清晰的核內(nèi),因不同品系小鼠原核大小和核膜清晰度差異顯著,致轉(zhuǎn)基因效率受到所選小鼠品系的影響,而慢病毒卵周隙注射時(shí),注射針無(wú)需插入核內(nèi),因而不受這一限制,并且操作較原核顯微注射法簡(jiǎn)單的多;(2)慢病毒卵周隙注射時(shí)對(duì)細(xì)胞膜和核膜均無(wú)損傷,因而胚胎存活率高;(3)慢病毒載體法的轉(zhuǎn)基因效率遠(yuǎn)遠(yuǎn)高于傳統(tǒng)的原核顯微注射法。 掌握慢病毒載體法制備轉(zhuǎn)基因小鼠技術(shù)的現(xiàn)時(shí)重要性和必要性利用遺傳工程小鼠在體內(nèi)解析基因功能的策略可分為:基因功能缺失(Loss of genefunction)和基因功能獲得(Gain of gene function)。在體內(nèi)實(shí)現(xiàn)基因功能缺失的主要策略主要包括:(條件性)基因敲除、(條件性)轉(zhuǎn)基因RNA干涉(RNAi)和負(fù)顯性突變體(Dominant negative mutant)技術(shù)等,而在體內(nèi)實(shí)現(xiàn)基因功能獲得的主要策略:目的基因(條件性)過(guò)表達(dá)等。近若干年來(lái),針對(duì)不同基因采用不同的基因功能研究策略,利用不同類(lèi)型的慢病毒載體借助慢病毒載體法建立了相應(yīng)的轉(zhuǎn)基因小鼠,在轉(zhuǎn)基因小鼠體內(nèi)已實(shí)現(xiàn)或有望實(shí)現(xiàn)如下目標(biāo):(1)基因功能獲得[目的基因(條件性)過(guò)表達(dá)]和(2)基因功能缺失[(條件性)轉(zhuǎn)基因RNAi和負(fù)顯性突變體技術(shù)],以上策略和方法許多已成功用于在小鼠體內(nèi)解析基因功能,這些都說(shuō)明基于慢病毒載體法制備的轉(zhuǎn)基因小鼠為活體內(nèi)研究基因功能提供了理想的三維研究體系。 靈敏的非損傷、實(shí)時(shí)和可視化體內(nèi)示蹤移植供體細(xì)胞及其在體內(nèi)衍生的細(xì)胞目前,除胚胎干細(xì)胞外,小鼠不同組織來(lái)源的成體干細(xì)胞均未建立成熟的體外培養(yǎng)體系,這給外源基因(包括報(bào)告基因)導(dǎo)入帶來(lái)諸多不便;為此,在進(jìn)行移植實(shí)驗(yàn)時(shí),往往從報(bào)告基因轉(zhuǎn)基因小鼠(報(bào)告基因的表達(dá)受普遍性的/無(wú)細(xì)胞或無(wú)組織特異的啟動(dòng)子調(diào)控)相應(yīng)組織(如骨髓等)中獲取報(bào)告基因標(biāo)記的待移植細(xì)胞�；铙w內(nèi)生物發(fā)光[用熒光素酶(Luciferase,Luc)基因標(biāo)記細(xì)胞等)成像和熒光[熒光報(bào)告基團(tuán)(GFP和RFP等)]成像各有其優(yōu)點(diǎn),如生物發(fā)光成像靈敏度高、特異性極強(qiáng)、可精確定量和組織穿透能力強(qiáng)等,而熒光成像雖有背景噪音、靈敏度低和穿透能力弱等缺點(diǎn),但熒光信號(hào)強(qiáng)、標(biāo)記靶點(diǎn)多樣、觀察直觀和檢測(cè)方便等。因此,對(duì)于不同研究,應(yīng)根據(jù)生物發(fā)光成像和熒光成像各自特點(diǎn)和優(yōu)勢(shì)進(jìn)行選擇。為將生物發(fā)光和熒光的優(yōu)點(diǎn)集為一身,有研究者通過(guò)GFP轉(zhuǎn)基因小鼠和Luc轉(zhuǎn)基因小鼠交配以獲得雙報(bào)告基因(GFP和Luc)轉(zhuǎn)基因小鼠,并進(jìn)而從其骨髓中分離得到雙報(bào)告基因標(biāo)記的移植供體細(xì)胞——造血干細(xì)胞(HSCs),以供移植實(shí)驗(yàn)來(lái)研究造血重構(gòu),從而實(shí)現(xiàn)非損傷、實(shí)時(shí)和可視化體內(nèi)監(jiān)測(cè)造血重構(gòu)過(guò)程。目前,報(bào)告基因(GFP、mRFP或Luc)表達(dá)受無(wú)細(xì)胞或無(wú)組織特異啟動(dòng)子驅(qū)動(dòng)的轉(zhuǎn)基因小鼠均是單報(bào)告基因的,而通過(guò)以上方法獲得雙報(bào)告基因標(biāo)記的供體移植細(xì)胞需花費(fèi)較長(zhǎng)時(shí)間。 有鑒于此,本課題擬借助慢病毒載體法制備雙報(bào)告基因Fluc-mRFP(即螢火蟲(chóng)熒光素酶和單體紅色熒光蛋白)轉(zhuǎn)基因小鼠,以實(shí)現(xiàn)下列目標(biāo):(1)熟練掌握基于慢病毒載體法制備轉(zhuǎn)基因小鼠的技術(shù);(2)建立雙報(bào)告基因Fluc-mRFP轉(zhuǎn)基因小鼠,以為后續(xù)研究(如干細(xì)胞在腫瘤發(fā)生、發(fā)展和轉(zhuǎn)移中的作用和造血重構(gòu)等)提供雙報(bào)告基因標(biāo)記的各種移植用供體細(xì)胞,以借助此類(lèi)三報(bào)告基因標(biāo)記的供體細(xì)胞將生物發(fā)光(Bioluminescence)和熒光(Fluorescence)兩項(xiàng)技術(shù)融為一身,從而為實(shí)現(xiàn)靈敏的非損傷、實(shí)時(shí)和可視化體內(nèi)示蹤移植供體細(xì)胞及其在體內(nèi)衍生的細(xì)胞奠定基礎(chǔ)。 目的: 基于慢病毒介導(dǎo)的轉(zhuǎn)基因方法制備Fluc-mRFP(FR)轉(zhuǎn)基因小鼠,即通過(guò)建立FR轉(zhuǎn)基因小鼠得以掌握慢病毒載體法制備轉(zhuǎn)基因小鼠的技術(shù),從而實(shí)現(xiàn)多種目的(見(jiàn)上)。 方法: 1)慢病毒載體phUb-FR鑒定 酶切鑒定分別使用NotⅠ、MluⅠ和BamHⅠ對(duì)phUb-FR進(jìn)行單酶切,酶切產(chǎn)物進(jìn)行1%瓊脂糖凝膠電泳。 PCR鑒定根據(jù)FR序列設(shè)計(jì)引物分別擴(kuò)增Flue、mRFP和ttk基因;PCR擴(kuò)增后,取5μl反應(yīng)液進(jìn)行2%瓊脂糖凝膠電泳。 2)慢病毒生產(chǎn)與鑒定 慢病毒包裝與濃縮按標(biāo)準(zhǔn)程序進(jìn)行慢病毒包裝(脂質(zhì)體介導(dǎo)的瞬時(shí)轉(zhuǎn)染)、超速離心濃縮和保存等。 慢病毒成功生產(chǎn)的鑒定用病毒上清或濃縮后的病毒感染293FT細(xì)胞,24～48h后熒光顯微鏡下觀察是否見(jiàn)紅色熒光。 3)利用細(xì)胞模型在體外驗(yàn)證Flue和mRFP能否正常表達(dá) 從正常293FT細(xì)胞和病毒感染后的293FT細(xì)胞提取總RNA,并進(jìn)而采用RT-PCR檢測(cè)Fluc和mRFP能否正常表達(dá)。 4)慢病毒載體法制備FR轉(zhuǎn)基因小鼠 特殊用途小鼠準(zhǔn)備按標(biāo)準(zhǔn)程序準(zhǔn)備受精卵供體母鼠、種公鼠、結(jié)扎公鼠和假孕母鼠等; 慢病毒卵周隙注射制備FR轉(zhuǎn)基因小鼠將濃縮后的病毒注射入小鼠受精卵的卵周隙中,然后將注射后狀態(tài)良好的受精卵移植進(jìn)ICR假孕母鼠輸卵管內(nèi),并將假孕母鼠送回籠內(nèi)并觀察直至恢復(fù)知覺(jué),仔鼠一般19.5～20.0d后出生。 5) FR轉(zhuǎn)基因首建鼠的篩選與鑒定 獲得子代小鼠后,首先應(yīng)用體視熒光顯微鏡、小動(dòng)物活體成像儀和流式細(xì)胞儀等檢測(cè)各組織中mRFP和Fluc表達(dá),并應(yīng)用PCR技術(shù)鑒定轉(zhuǎn)基因FR是否成功整合進(jìn)小鼠基因組,來(lái)進(jìn)一步驗(yàn)證上述結(jié)果,以獲得FR轉(zhuǎn)基因小鼠,即獲得FR轉(zhuǎn)基因首建鼠。 (1)體視熒光顯微鏡和小動(dòng)物活體成像儀檢測(cè)mRFP和Flue表達(dá)以篩選和鑒定FR轉(zhuǎn)基因鼠: 體視熒光顯微鏡初篩FR轉(zhuǎn)基因鼠將出生幾天后的乳鼠置于體視熒光顯微鏡下,檢測(cè)mRFP表達(dá)以初步鑒定FR轉(zhuǎn)基因鼠,并從中初篩出熒光強(qiáng)度適中的乳鼠。 體視熒光顯微鏡檢測(cè)轉(zhuǎn)基因鼠各器官mRFP表達(dá)在小鼠出生3周后,小鼠灌注后取出各臟器,并熒光鏡檢,方法如下:小鼠深度麻醉后,用PBS自小鼠左心室注入至右心房流出,直至流出液變成無(wú)色透明后,收集主要臟器(如心臟、肺、腦、腎臟、肝臟、脾臟、胸腺、肌肉、皮膚、小腸等)。并在熒光體視顯微鏡下檢測(cè)紅色熒光。 小動(dòng)物活體成像儀檢測(cè)Flue表達(dá)檢測(cè)前,小鼠腹腔注射D-luciferin(0.15mg/g body wt),同時(shí)麻醉小鼠;注射底物5-10 min后,利用小動(dòng)物活體成像儀檢測(cè)Fluc表達(dá)。 (2) PCR檢測(cè)小鼠基因組中Flu-mRFP整合 以從潛在的FR轉(zhuǎn)基因小鼠和野生型小鼠(陰性對(duì)照)鼠尾組織提取的基因組DNA及質(zhì)粒phUb-FR為模板,分別PCR擴(kuò)增Flu、mRFP和ttk基因片段,以鑒定潛在的FR轉(zhuǎn)基因小鼠的基因型。 6) FR轉(zhuǎn)基因首建鼠繁殖傳代及轉(zhuǎn)基因遺傳和表達(dá)穩(wěn)定性檢測(cè) 將mRFP表達(dá)陽(yáng)性及PCR陽(yáng)性的首建鼠與野生型ICR鼠交配以傳代,獲得F_1后,用體視熒光顯微鏡,檢測(cè)mRFP是否在F_1代表達(dá),對(duì)其表達(dá)穩(wěn)定性做出判斷,并進(jìn)而間接判斷轉(zhuǎn)基因是否穩(wěn)定遺傳。 結(jié)果: 1)慢病毒載體phUb-FR鑒定 酶切鑒定phUb-FR經(jīng)NotⅠ、MluⅠ和BamHⅠ分別單酶切,酶切產(chǎn)物經(jīng)1%瓊脂糖凝膠電泳均可見(jiàn)兩條預(yù)期大小的條帶。 PCR鑒定以質(zhì)粒phUb-FR為模板,分別擴(kuò)增Fluc、mRFP、ttk,三PCR產(chǎn)物大小均與預(yù)期值相符。 2)慢病毒包裝、濃縮及鑒定 攜帶FR基因慢病毒的生產(chǎn)與鑒定將phUb-FR與病毒包裝質(zhì)粒共轉(zhuǎn)染293FT細(xì)胞,48h后倒置熒光顯微鏡下可見(jiàn)紅色熒光,預(yù)示轉(zhuǎn)染成功。用收集的病毒上清感染293FT細(xì)胞,48h后倒置熒光顯微鏡下可觀察到紅色熒光,這進(jìn)一步說(shuō)明病毒已成功生產(chǎn);同時(shí)也表明轉(zhuǎn)基因mRFP能夠正常表達(dá)。 3)利用細(xì)胞模型在體外驗(yàn)證Flue和mRFP能否正常表達(dá) 用攜帶FR慢病毒感染293FT細(xì)胞,48h后在倒置熒光顯微鏡下見(jiàn)紅色熒光,這表明轉(zhuǎn)基因mRFP能夠正常表達(dá);同時(shí),利用RT-PCR在mRNA水平亦證實(shí)轉(zhuǎn)基因Fluc和mRFP能夠正常表達(dá)。 4) FR轉(zhuǎn)基因小鼠的建立 慢病毒注射入580枚單細(xì)胞受精卵的卵周隙,存活胚胎556枚,將525枚卵周隙注射有慢病毒的胚胎移植給28只假孕母鼠,22只懷孕,共獲仔鼠136只,DNA水平檢測(cè)證實(shí)其中63只基因組中整合有外源基因Flu、mRFP和ttk,即獲得63只PCR陽(yáng)性的FR轉(zhuǎn)基因首建鼠,首建鼠FR整合率達(dá)46%。在蛋白水平,利用體視熒光顯微鏡和/或小動(dòng)物活體成像系統(tǒng)檢測(cè)mRFP表達(dá),證實(shí)63只PCR陽(yáng)性的轉(zhuǎn)基因小鼠中,47只正常表達(dá)mRFP,其中16只強(qiáng)表達(dá)mRFP,31只弱表達(dá)mRFP。同時(shí),利用小動(dòng)物活體成像儀亦可在mRFP陽(yáng)性的FR轉(zhuǎn)基因小鼠檢測(cè)到Fluc表達(dá)。 5) FR首建鼠繁殖傳代及外源基因遺傳和表達(dá)穩(wěn)定性檢測(cè) 體視熒光顯微鏡檢測(cè)顯示,F_1代鼠中部分個(gè)體表達(dá)mRFP,這預(yù)示外源基因不僅可以從一代向下一代穩(wěn)定傳遞,且能夠穩(wěn)定表達(dá)。 6) FR轉(zhuǎn)基因鼠主要臟器mRFP表達(dá)的檢測(cè) 在體視熒光顯微鏡下,F1代的FR轉(zhuǎn)基因小鼠的腦、脾臟、腎臟和小腸等可見(jiàn)紅色熒光。 結(jié)論: 1)運(yùn)用慢病毒載體法成功建立FR轉(zhuǎn)基因小鼠,獲得63只PCR陽(yáng)性首建鼠,首建鼠FR整合率達(dá)46%; 2) 63只PCR陽(yáng)性首建鼠中,47只正常表達(dá)mRFP,其中16只強(qiáng)表達(dá)mRFP;同時(shí)也可在PCR陽(yáng)性首建鼠上檢測(cè)到Fluc表達(dá); 3) FR轉(zhuǎn)基因首建鼠攜帶的外源基因不僅可以向下一代傳遞,且FR轉(zhuǎn)基因能夠穩(wěn)定表達(dá); 4) FR轉(zhuǎn)基因鼠的主要臟器(如腦、脾臟、腎臟和小腸等)可見(jiàn)紅色熒光。
[Abstract]:The functional mice of genetic engineering have become the most important and best model experimental animals for studying gene function, developing biological research, building animal models of human diseases and studying the pathogenesis of human diseases. The most effective way to identify gene function is to observe gene function loss or to observe gene function deficiency. Genetic engineering mice (Genetically modified mice, GMMs) produced by genetically modified (Transgene) and gene targeting (Gene targeting) and gene knocking (Geneknockin) are one of the most valuable tools. In order to study the development of human body at the whole animal level, analyze the function of gene function or disease related gene, study the pathogenesis of human disease, solve the susceptibility of certain disease, identify the drug target, the screening of new drug and the judgment of curative effect.
The predominant prokaryotic microinjection method for the preparation of transgenic mice by the lentivirus carrier method is the most classic and most mature method for preparing transgenic mice. However, the efficiency of the microinjection preparation of transgenic animals is very low. In recent years, with the advent of different uses of the lentivirus vector, the lentivirus mediated transgenic animal production side So far, the use of lentivirus vector has successfully prepared transgenic mice, rats, pigs, cattle and chickens. The efficiency of this method is much higher than that of the previous method. This method has been proved to be widely used in the production of transgenic animals from mammalian to poultry. The following advantages are as follows: (1) the injection needle must be inserted into a clear nucleus of the nuclear membrane at the time of prokaryotic microinjection, due to the significant difference in the size of the nucleus and the clarity of the nuclear membrane in different strains of mice, resulting in the effect of the transgenic efficiency on the selected mice, and the injection needles do not need to be inserted into the nucleus when the lentivirus egg gap is injected, and the operation is not limited. The prokaryotic microinjection method is simple. (2) there is no damage to the cell membrane and the nuclear membrane during the injection of the lentivirus, so the survival rate of the embryo is high. (3) the efficiency of the lentivirus vector method is far higher than the traditional prokaryotic microinjection method.
The current importance and necessity of using the lentivirus vector method to prepare transgenic mice can be divided into Loss of genefunction and Gain of gene function. The main strategies for the loss of gene function in vivo include: ( Conditionality) gene knockout, (conditioned) transgenic RNA interference (RNAi) and negative dominant mutant (Dominant negative mutant) technology, and the main strategy to achieve gene function in the body: the target gene (conditioned) overexpression. In recent years, different types of gene function research strategies have been used for different genes to use different types of genes. The lentivirus vector has established the corresponding transgenic mice with the aid of the lentivirus vector, which has been realized in transgenic mice or is expected to achieve the following objectives: (1) gene function acquisition [target gene (conditioned) overexpression] and (2) gene function deletion [(conditioned) transgenic RNAi and negative dominant mutant technique], many of the strategies and methods have been done It was successfully used to analyze gene function in mice, which indicated that transgenic mice based on the lentivirus vector method provided an ideal three-dimensional research system for the study of gene function in living in vivo.
In addition to the embryonic stem cells, the adult stem cells from different tissues of mice have not established a mature culture system in vitro, which brings many inconveniences to the exogenous gene (including the reporter gene). Therefore, the transplantation experiment is carried out. At the time, the transplanted cells are often obtained from the reporter gene transgenic mice (the expression of the reporter gene is regulated by the universal / acellular or unorganized specific promoter) in the corresponding tissue (such as bone marrow). In vivo bioluminescence [using Luciferase (Luc) gene labeled cells, etc.) imaging and fluorescence [fluorescence report] The imaging of groups (GFP and RFP) have their own advantages, such as high sensitivity, strong specificity, accurate quantification and strong tissue penetration ability, while fluorescence imaging has the disadvantages of background noise, low sensitivity and weak penetration ability, but the fluorescence signal is strong, the target points are varied, the observation is intuitionistic and the detection is convenient. The study should be selected according to the characteristics and advantages of bioluminescence imaging and fluorescence imaging. In order to combine the advantages of bioluminescence and fluorescence, the researchers copated by GFP transgenic mice and Luc transgenic mice to obtain the double reporter gene (GFP and Luc) transgenic mice and then separate the double reporter gene from the bone marrow. The transplanted donor cells, hematopoietic stem cells (HSCs), are used to study hematopoietic reconfiguration in order to achieve non injury, real-time and visual monitoring of hematopoietic reconfiguration in vivo. At present, the reported gene (GFP, mRFP or Luc) expression of transgenic mice driven by non cell or unorganized specific promoters is a single reporter gene. These methods can take longer time to obtain donor cells transplanted with double report genes.
In view of this, we intend to prepare transgenic mice with double reporter gene Fluc-mRFP (fluorescent firefly luciferase and mono red fluorescent protein) by the lentivirus vector method, in order to achieve the following objectives: (1) mastering the technique of preparing transgenic mice based on the lentivirus vector method; (2) to establish a double reporter gene transgenic mice. Further studies (such as the role of stem cells in tumorigenesis, development and metastasis and hematopoiesis) provide a variety of transplant donor cells with double reporting gene markers, in order to achieve sensitivity by combining the two techniques of Bioluminescence and fluorescence (Fluorescence) with the donor cells of such three reported gene markers. Non injury, real-time and visualized tracing of transplanted donor cells and their derived cells in vivo lay the foundation.
Objective:
Fluc-mRFP (FR) transgenic mice were prepared based on the lentivirus mediated transgenic method, that is, by establishing the FR transgenic mice to master the technique of the lentivirus vector preparation of transgenic mice, so as to achieve a variety of purposes (see).
Method:
1) identification of lentivirus vector phUb-FR
Enzyme digestion identification was performed on Not, Mlu I and BamH I by single enzyme digestion of phUb-FR and 1% agarose gel electrophoresis.
PCR identified primers to amplify Flue, mRFP and TTK genes according to FR sequences. After PCR amplification, 2% l agar gel electrophoresis was carried out with 5 L reaction solution.
2) production and identification of lentivirus
Lentivirus packaging and concentration were carried out according to standard procedures for lentivirus packaging (liposome mediated transient transfection), ultracentrifugation and preservation.
The identification of successful production of lentivirus infected 293FT cells with virus supernatant or concentrated virus. Red fluorescence was observed after 24 to 48h fluorescence microscope.
3) using cell models to verify the normal expression of Flue and mRFP in vitro.
The total RNA was extracted from normal 293FT cells and 293FT cells after virus infection, and then RT-PCR was used to detect whether Fluc and mRFP could express normally.
4) preparation of FR transgenic mice by lentivirus vector method
Special purpose mice are going to prepare fertilized egg donor mothers, male rats, male rats and pseudo pregnant rats according to standard procedures.
FR transgenic mice were injected into the egg gap of the fertilized egg of mice by injection of the lentivirus, and then the fertilized eggs were transplanted into the fallopian tube of ICR pregnant mice, and the pregnant mice were sent back to the cage and observed until they were recovered. The offspring were born after 19.5 to 20.0d.
5) screening and identification of FR transgenic first mice
After obtaining the offspring, the expression of mRFP and Fluc in each tissue was detected by stereoscopic fluorescence microscope, small animal living imaging instrument and flow cytometry, and PCR technique was used to identify whether genetically modified FR was successfully integrated into the mouse genome to further verify the above results in order to obtain the FR transgenic mice, that is, to obtain FR transgenic first mice.
(1) stereoscopic fluorescence microscope and small animal imaging system were used to detect mRFP and Flue expression to screen and identify FR transgenic mice:
A stereoscopic fluorescent microscope was used to screen FR transgenic mice after a few days after birth. The expression of mRFP was detected to identify the FR transgenic mice and to screen out the milk mice with moderate fluorescence intensity.
The expression of mRFP in all organs of transgenic mice was detected by a stereoscopic fluorescence microscope after 3 weeks of birth. After the mice were perfused, the organs were taken out and examined by fluorescence microscopy. The methods were as follows: after the deep anesthesia, the mice were injected into the right atrium from the left ventricle of the mice with PBS until the effluent became colorless and transparent, and the main organs (such as the heart, lungs, brain, kidneys, liver, and liver) were collected. Spleen, thymus, muscle, skin, small intestine, etc.) and red fluorescence was detected under fluorescence microscope.
The mice were intraperitoneally injected with D-luciferin (0.15mg/g body wt), and the mice were anesthetized at the same time before the Flue expression was detected by the small animal living imaging instrument. After the injection of substrate 5-10 min, the expression of Fluc was detected by the small animal living imager.
(2) PCR detection of Flu-mRFP integration in the genome of mice
The genomic DNA and plasmid phUb-FR extracted from the tail tissues of the potential FR transgenic mice and the wild mice (negative control) were used as templates to amplify the Flu, mRFP and TTK fragments of the Flu, mRFP and TTK genes to identify the genotypes of the potential FR transgenic mice.
6) breeding and passage of FR transgenic mice and detection of transgene expression and stability.
After mating the mRFP positive and PCR positive first mice with the wild ICR mice to pass the generation, the F_1 was obtained, and the stereoscopic fluorescence microscope was used to determine whether the mRFP was in the F_1 representation. The stability of the expression was judged, and then the genetic stability of the gene was indirectly judged.
Result:
1) identification of lentivirus vector phUb-FR
Enzyme digestion identified phUb-FR by single enzyme digestion through Not I, Mlu I and BamH I, and two expected size bands were obtained from the products of digestion by 1% agarose gel electrophoresis.
PCR identified plasmid phUb-FR as template and amplified Fluc, mRFP, TTK, and three PCR respectively, and the product size was consistent with the expected value.
2) packaging, concentration and identification of lentivirus
The production and identification of FR gene lentivirus carried phUb-FR and virus packaging plasmids into 293FT cells. After 48h inverted fluorescence microscopy, red fluorescence could be seen, which indicated that the transfection was successful. 293FT cells were infected with the collected virus supernatant, and the red fluorescence could be observed under the inverted fluorescence microscope after 48h, which further indicated that the virus had been produced successfully. It also indicates that the transgenic mRFP can be expressed normally.
3) using cell models to verify the normal expression of Flue and mRFP in vitro.
293FT cells were infected with FR lentivirus, and red fluorescence was observed under inverted fluorescence microscope after 48h, which indicated that transgenic mRFP could be expressed normally. At the same time, the normal expression of genetically modified Fluc and mRFP was confirmed by RT-PCR at the level of mRNA.
4) establishment of FR transgenic mice
The lentivirus was injected into the ovum gap of 580 single cell fertilized eggs, and 556 embryos survived, and 525 eggs with lentivirus were transplanted to 28 pregnant mice, 22 pregnant and 136 offspring were obtained. The DNA level test confirmed that the 63 genome was integrated with the exogenous gene Flu, mRFP and TTK, that is, 63 PCR positive FR GM heads were obtained. The FR integration rate of the first rat was 46%. at the protein level, and the expression of mRFP was detected by stereoluminescence microscope and / or small animal living imaging system, and 47 of the 63 PCR positive transgenic mice expressed mRFP, of which 16 strongly expressed mRFP, 31 weak expressed mRFP. simultaneously, and the small animal living imager could also be in mRFP positive FR. Transgene
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R346

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