慢病毒载体法制备双报告基因Fluc-mRFP转基因小鼠

发布时间：2018-06-21 20:36

本文选题：慢病毒载体法 + 转基因小鼠　；参考：《南方医科大学》2008年硕士论文

【摘要】： 遗传工程小鼠的功用小鼠因其一些独特特点和优势已成为研究基因功能、开展发育生物学研究、建造人类疾病动物模型及研究人类疾病发病机制等的最重要的和最佳的模式实验动物。识别基因功能最有效方法是观察基因功能缺失或基因过表达后,细胞和机体所产生的表型变化。利用转基因(Transgene)和基因打靶(Gene targeting)[包括基因敲除(Gene knockout)和基因敲入(Geneknockin)等]等技术产生的遗传工程小鼠(Genetically modified mice,GMMs)是最具研究价值的工具之一。GMMs已成为在整体动物水平研究人体发育、解析基因功能或疾病相关基因功能,研究人类疾病发病机制,解答特定人群对某种疾病的易感性,识别药靶,新药筛选和疗效判定等最为有效的手段。慢病毒载体法制备转基因小鼠的优势原核显微注射法为目前最经典的和最成熟制备转基因小鼠的方法,被广泛采用,但是显微注射制备转基因动物的效率很低。近年来,随着不同用途慢病毒载体的问世,慢病毒介导的转基因动物制作方法逐渐受到人们青睐;迄今,利用慢病毒载体法已成功制备了转基因小鼠、大鼠、猪、牛和鸡等,效率较以往的方法高许多,该方法已被证明是普遍适用于从哺乳类动物到禽类的转基因动物制作方法。与原核显微注射法相比,慢病毒载体法有如下优势:(1)原核显微注射时注射针必须插入核膜清晰的核内,因不同品系小鼠原核大小和核膜清晰度差异显著,致转基因效率受到所选小鼠品系的影响,而慢病毒卵周隙注射时,注射针无需插入核内,因而不受这一限制,并且操作较原核显微注射法简单的多;(2)慢病毒卵周隙注射时对细胞膜和核膜均无损伤,因而胚胎存活率高;(3)慢病毒载体法的转基因效率远远高于传统的原核显微注射法。掌握慢病毒载体法制备转基因小鼠技术的现时重要性和必要性利用遗传工程小鼠在体内解析基因功能的策略可分为:基因功能缺失(Loss of genefunction)和基因功能获得(Gain of gene function)。在体内实现基因功能缺失的主要策略主要包括:(条件性)基因敲除、(条件性)转基因RNA干涉(RNAi)和负显性突变体(Dominant negative mutant)技术等,而在体内实现基因功能获得的主要策略:目的基因(条件性)过表达等。近若干年来,针对不同基因采用不同的基因功能研究策略,利用不同类型的慢病毒载体借助慢病毒载体法建立了相应的转基因小鼠,在转基因小鼠体内已实现或有望实现如下目标:(1)基因功能获得[目的基因(条件性)过表达]和(2)基因功能缺失[(条件性)转基因RNAi和负显性突变体技术],以上策略和方法许多已成功用于在小鼠体内解析基因功能,这些都说明基于慢病毒载体法制备的转基因小鼠为活体内研究基因功能提供了理想的三维研究体系。灵敏的非损伤、实时和可视化体内示踪移植供体细胞及其在体内衍生的细胞目前,除胚胎干细胞外,小鼠不同组织来源的成体干细胞均未建立成熟的体外培养体系,这给外源基因(包括报告基因)导入带来诸多不便;为此,在进行移植实验时,往往从报告基因转基因小鼠(报告基因的表达受普遍性的/无细胞或无组织特异的启动子调控)相应组织(如骨髓等)中获取报告基因标记的待移植细胞。活体内生物发光[用荧光素酶(Luciferase,Luc)基因标记细胞等)成像和荧光[荧光报告基团(GFP和RFP等)]成像各有其优点,如生物发光成像灵敏度高、特异性极强、可精确定量和组织穿透能力强等,而荧光成像虽有背景噪音、灵敏度低和穿透能力弱等缺点,但荧光信号强、标记靶点多样、观察直观和检测方便等。因此,对于不同研究,应根据生物发光成像和荧光成像各自特点和优势进行选择。为将生物发光和荧光的优点集为一身,有研究者通过GFP转基因小鼠和Luc转基因小鼠交配以获得双报告基因(GFP和Luc)转基因小鼠,并进而从其骨髓中分离得到双报告基因标记的移植供体细胞——造血干细胞(HSCs),以供移植实验来研究造血重构,从而实现非损伤、实时和可视化体内监测造血重构过程。目前,报告基因(GFP、mRFP或Luc)表达受无细胞或无组织特异启动子驱动的转基因小鼠均是单报告基因的,而通过以上方法获得双报告基因标记的供体移植细胞需花费较长时间。有鉴于此,本课题拟借助慢病毒载体法制备双报告基因Fluc-mRFP(即萤火虫荧光素酶和单体红色荧光蛋白)转基因小鼠,以实现下列目标:(1)熟练掌握基于慢病毒载体法制备转基因小鼠的技术;(2)建立双报告基因Fluc-mRFP转基因小鼠,以为后续研究(如干细胞在肿瘤发生、发展和转移中的作用和造血重构等)提供双报告基因标记的各种移植用供体细胞,以借助此类三报告基因标记的供体细胞将生物发光(Bioluminescence)和荧光(Fluorescence)两项技术融为一身,从而为实现灵敏的非损伤、实时和可视化体内示踪移植供体细胞及其在体内衍生的细胞奠定基础。目的: 基于慢病毒介导的转基因方法制备Fluc-mRFP(FR)转基因小鼠,即通过建立FR转基因小鼠得以掌握慢病毒载体法制备转基因小鼠的技术,从而实现多种目的(见上)。方法: 1)慢病毒载体phUb-FR鉴定酶切鉴定分别使用NotⅠ、MluⅠ和BamHⅠ对phUb-FR进行单酶切,酶切产物进行1%琼脂糖凝胶电泳。 PCR鉴定根据FR序列设计引物分别扩增Flue、mRFP和ttk基因;PCR扩增后,取5μl反应液进行2%琼脂糖凝胶电泳。 2)慢病毒生产与鉴定慢病毒包装与浓缩按标准程序进行慢病毒包装(脂质体介导的瞬时转染)、超速离心浓缩和保存等。慢病毒成功生产的鉴定用病毒上清或浓缩后的病毒感染293FT细胞,24～48h后荧光显微镜下观察是否见红色荧光。 3)利用细胞模型在体外验证Flue和mRFP能否正常表达从正常293FT细胞和病毒感染后的293FT细胞提取总RNA,并进而采用RT-PCR检测Fluc和mRFP能否正常表达。 4)慢病毒载体法制备FR转基因小鼠特殊用途小鼠准备按标准程序准备受精卵供体母鼠、种公鼠、结扎公鼠和假孕母鼠等; 慢病毒卵周隙注射制备FR转基因小鼠将浓缩后的病毒注射入小鼠受精卵的卵周隙中,然后将注射后状态良好的受精卵移植进ICR假孕母鼠输卵管内,并将假孕母鼠送回笼内并观察直至恢复知觉,仔鼠一般19.5～20.0d后出生。 5) FR转基因首建鼠的筛选与鉴定获得子代小鼠后,首先应用体视荧光显微镜、小动物活体成像仪和流式细胞仪等检测各组织中mRFP和Fluc表达,并应用PCR技术鉴定转基因FR是否成功整合进小鼠基因组,来进一步验证上述结果,以获得FR转基因小鼠,即获得FR转基因首建鼠。 (1)体视荧光显微镜和小动物活体成像仪检测mRFP和Flue表达以筛选和鉴定FR转基因鼠: 体视荧光显微镜初筛FR转基因鼠将出生几天后的乳鼠置于体视荧光显微镜下,检测mRFP表达以初步鉴定FR转基因鼠,并从中初筛出荧光强度适中的乳鼠。体视荧光显微镜检测转基因鼠各器官mRFP表达在小鼠出生3周后,小鼠灌注后取出各脏器,并荧光镜检,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,直至流出液变成无色透明后,收集主要脏器(如心脏、肺、脑、肾脏、肝脏、脾脏、胸腺、肌肉、皮肤、小肠等)。并在荧光体视显微镜下检测红色荧光。小动物活体成像仪检测Flue表达检测前,小鼠腹腔注射D-luciferin(0.15mg/g body wt),同时麻醉小鼠;注射底物5-10 min后,利用小动物活体成像仪检测Fluc表达。 (2) PCR检测小鼠基因组中Flu-mRFP整合以从潜在的FR转基因小鼠和野生型小鼠(阴性对照)鼠尾组织提取的基因组DNA及质粒phUb-FR为模板,分别PCR扩增Flu、mRFP和ttk基因片段,以鉴定潜在的FR转基因小鼠的基因型。 6) FR转基因首建鼠繁殖传代及转基因遗传和表达稳定性检测将mRFP表达阳性及PCR阳性的首建鼠与野生型ICR鼠交配以传代,获得F_1后,用体视荧光显微镜,检测mRFP是否在F_1代表达,对其表达稳定性做出判断,并进而间接判断转基因是否稳定遗传。结果: 1)慢病毒载体phUb-FR鉴定酶切鉴定phUb-FR经NotⅠ、MluⅠ和BamHⅠ分别单酶切,酶切产物经1%琼脂糖凝胶电泳均可见两条预期大小的条带。 PCR鉴定以质粒phUb-FR为模板,分别扩增Fluc、mRFP、ttk,三PCR产物大小均与预期值相符。 2)慢病毒包装、浓缩及鉴定携带FR基因慢病毒的生产与鉴定将phUb-FR与病毒包装质粒共转染293FT细胞,48h后倒置荧光显微镜下可见红色荧光,预示转染成功。用收集的病毒上清感染293FT细胞,48h后倒置荧光显微镜下可观察到红色荧光,这进一步说明病毒已成功生产;同时也表明转基因mRFP能够正常表达。 3)利用细胞模型在体外验证Flue和mRFP能否正常表达用携带FR慢病毒感染293FT细胞,48h后在倒置荧光显微镜下见红色荧光,这表明转基因mRFP能够正常表达;同时,利用RT-PCR在mRNA水平亦证实转基因Fluc和mRFP能够正常表达。 4) FR转基因小鼠的建立慢病毒注射入580枚单细胞受精卵的卵周隙,存活胚胎556枚,将525枚卵周隙注射有慢病毒的胚胎移植给28只假孕母鼠,22只怀孕,共获仔鼠136只,DNA水平检测证实其中63只基因组中整合有外源基因Flu、mRFP和ttk,即获得63只PCR阳性的FR转基因首建鼠,首建鼠FR整合率达46%。在蛋白水平,利用体视荧光显微镜和/或小动物活体成像系统检测mRFP表达,证实63只PCR阳性的转基因小鼠中,47只正常表达mRFP,其中16只强表达mRFP,31只弱表达mRFP。同时,利用小动物活体成像仪亦可在mRFP阳性的FR转基因小鼠检测到Fluc表达。 5) FR首建鼠繁殖传代及外源基因遗传和表达稳定性检测体视荧光显微镜检测显示,F_1代鼠中部分个体表达mRFP,这预示外源基因不仅可以从一代向下一代稳定传递,且能够稳定表达。 6) FR转基因鼠主要脏器mRFP表达的检测在体视荧光显微镜下,F1代的FR转基因小鼠的脑、脾脏、肾脏和小肠等可见红色荧光。结论: 1)运用慢病毒载体法成功建立FR转基因小鼠,获得63只PCR阳性首建鼠,首建鼠FR整合率达46%; 2) 63只PCR阳性首建鼠中,47只正常表达mRFP,其中16只强表达mRFP;同时也可在PCR阳性首建鼠上检测到Fluc表达; 3) FR转基因首建鼠携带的外源基因不仅可以向下一代传递,且FR转基因能够稳定表达; 4) FR转基因鼠的主要脏器(如脑、脾脏、肾脏和小肠等)可见红色荧光。
[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
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

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