DKK1转基因体细胞克隆西藏小型猪的制备
发布时间：2018-08-21 13:59
【摘要】：研究背景和意义： 由于体型小、便于操作,而且解剖、生理生化特性与人特别相近的特点,小型猪已逐渐成为生物医药研究领域重要的实验动物,应用数量逐年大幅递增。 西藏小型猪是世界上体型较小的小型猪之一,来源于青藏高原、海拔2500-4300米的农区和办农牧区,是唯一能适应高海拔气候和以放牧为主的猪种,封闭的地理环境使西藏小型猪保存了非常纯正的品种资源。南方医科大学实验动物中心于2004年将西藏小型猪从西藏引进至广州进行实验动物化研究,目前已完成风土驯化及实验动物化研究,并开展了相关动物模型、药物试验及转基因克隆等研究。从免疫学、遗传学等研究发现,该品系具有其独特的免疫相关指标和遗传特征,加上其独特的外形,是一种优良的实验用小型猪品系。 小型猪的皮肤系统与人的有较大的相似性,故被认为是皮肤相关研究的理想模型猪和人的皮肤均由表皮、真皮、皮下组织三层组成；猪的皮肤在解剖结构、形态、生理生化特性与药理学等方面与人的非常相似,包括皮肤厚薄(如小型猪皮肤70-140微米厚,人皮肤50-120微米厚,而大鼠皮肤10-20微米厚)、皮肤形态学和增生动力学、皮肤修复再生性、烧伤皮肤的体液和代谢变化规律等。因此,猪被认为是皮肤和整形外科手术的标准模型,也是进行皮肤移植、化妆品安全性评价、紫外线照射、皮肤致癌、烧伤、冻伤、皮肤的老化及抗老化等研究的理想实验材料。但是,猪皮肤表面被毛,在对小型猪进行皮肤实验或外科手术之前,总是免不了繁琐的剃毛程序。 常见的无毛小型实验动物有裸鼠、豫医无毛小鼠和无毛豚鼠,其中无毛豚鼠已被广泛应用于皮肤相关研究(如生发剂疗效、皮肤过敏反应、植皮治疗、紫外线辐射反应等)。目前,世界上仅有的一种无毛猪品系为尤卡坦(Yucatan)小型猪,也叫墨西哥无毛猪,广泛应用于皮肤研究。目前,我国已培育了许多小型猪品系：包括五指山小型猪、贵州小型猪、广西巴马小型猪、版纳微型猪和西藏小型猪等,尚未培育出无毛小型猪品系,而生物医学研究急盼基于中国培育的小型猪品系创制无毛小型猪新品系。 创制无毛小型猪品系,将免除相关实验过程中繁杂的脱毛程序和由此造成的皮肤损伤,无毛小型猪可广泛用于皮肤移植、化妆品安全性评价、紫外线照射、皮肤致癌、烧伤、冻伤、皮肤老化及抗老化等实验,同时也为脱发研究提供了理想的大动物模型,对我国的生物医药研究有着重大意义。 通常可通过如下方法获得一种新的实验动物品系(如白花或无毛)：从自然出生的动物中筛选出具有突变表型的个体,然后进行近交繁育,最终获得具有此表型的新品系,前面提及的豫医无毛小鼠和无毛豚鼠都是通过这种方法培育的。但是对于大型实验动物来讲,通过如上方法获得具有突变表型的个体,并进而培育成新的品系,是非常困难的和不太现实的。 已有研究发现,皮肤特异性过表达小鼠DKK1转基因(其表达受人皮肤特异性的K14启动子调控)的转基因小鼠皮肤无毛(即获得了无毛小鼠),这是由于皮肤特异性过表达DKK1转基因导致小鼠毛发发育受阻,而该转基因小鼠的其它方面情况正常。大量研究表明,在人和高等动物(如小鼠和猪等)的发育过程中,不同物种间的同一基因(如DKK1)的编码序列高度同源,这决定其功能在不同物种间往往是保守的。猪DKK1基因的编码序列与人和小鼠的高度同源,这些预示DKK1基因的功能在人、猪和小鼠等间亦是保守的(即功能相同),这提示在转基因猪的皮肤特异性过表达猪DKK1转基因,亦有可能导致该DKK1转基因猪毛发发育受阻而变得无毛,从而获得无毛猪。 此外,采用慢病毒载体法制备了皮肤特异性表达绿色荧光蛋白(GFP)基因(GFP表达受人皮肤特异性的K14启动子调控)的转基因猪,证实人K14启动子在猪上能正常工作,且GFP基因的表达被限定在猪皮肤,而其它组织无GFP表达,这预示人K14启动子能控制其所调控的转基因在猪皮肤组织特异性表达。 目前,制备转基因猪的方法主要有两种：体细胞核移植法和慢病毒载体法,其中后一种方法制备转基因猪受制于受精卵采集和数量的限制,不能被普遍应用。基于此,本课题将采用体细胞核移植法制备转基因克隆猪。 综上,本课题拟借助转基因体细胞核移植法,建立猪皮肤特异性过表达猪DKK1转基因(猪DKK1转基因的表达受人K14启动子调控)的转基因克隆小型猪(即制作DKK1转基因体细胞克隆西藏小型猪),以实现在转基因小型猪皮肤特异性过表达DKK1转基因,进而导致转基因小型猪毛发发育受阻而变得无毛,从而实现创制无毛小型猪新品系的目标。 本课题拟首先基于本中心培育的西藏小型猪创制无毛小型猪,如获成功将进一步基于中国培育的其它小型猪品系采用同样的方法创制无毛小型猪新品系,以便更好满足多方面的使用需求。 方法： 1.构建携带西藏小型猪DKK1基因的慢病毒载体pERKDZG 首先从西藏小型猪肝脏组织提取RNA,逆转录为cDNA,设计引物并PCR扩增西藏小型猪DKK1(简写为pDKK1)基因编码区。然后通过酶切、in-fusion克隆的方法,进行如下3步DNA克隆： ①将pDKK1基因插入载体pK14-DKK1,替换其中的鼠DKK1片段,以O获得载体pK14-pDKK1; ②从载体pCDH-CMV-MCS-EF1a-RFP中扩增EF1a-RFP片段,插入至慢病毒载体pHAGE-fullEF1a-MCS-IzsGreen,获得载体pEREZG; ③从载体、pKl4-pDKK1中扩增K14-pDKK1片段,插入载体pEREZG,获得最终慢病毒载体pERKDZG。 2.借助慢病毒将目的转基因导入西藏小型猪胚胎成纤维细胞(PEFs) 利用慢病毒载体pERKDZG包装病毒,然后用携带目的转基因ERKDZG的慢病毒感染PEFs,借助RFP和GFP监测外源转基因导入与表达情况。对病毒感染后的PEFs进行扩大培养,然后借助流式细胞仪分选RFP阳性的细胞,最终获得纯的可用于体细胞核移植的经遗传修饰的PEFs,命名为PEF-DKK1。 从生产慢病毒时转染了pERKDZG的293T细胞提取总RNA,进行逆转录,RT-PCR检测细胞中DKK1基因表达。 3.K14启动子和转基因载体pERKDZG的体外功能验证 将pERKDZG分别转染皮肤细胞[即西藏小型猪皮肤成纤维细胞(PDFs)、小鼠皮肤黑色素瘤细胞系B-16]和非皮肤细胞[即PEFs、小鼠胚胎成纤维细胞(MEFs)、人鼻咽癌细胞系HNE1细胞、293T细胞],然后在倒置荧光显微镜下观察RFP和GFP在以上六种细胞中的表达情况,以验证K14启动子组织特异性和转基因载体pERKDZG的功能性。 4.通过体细胞核移植技术制作DKK1转基因体细胞克隆西藏小型猪 从屠宰场收集猪卵巢,实验室收集卵母细胞,并进行成熟培养。成熟培养40小时后,透明质酸酶处理,脱去卵丘,显微操作仪下进行卵母细胞的去核,然后将PEF-DKK1细胞注射入去核卵母细胞透明带下,然后电融合激活,使重构卵开始发育,体外短暂培养重构胚胎,将2-4细胞期的早期重构胚胎移植入发情母猪的输卵管,依赖代孕母猪生产转基因克隆西藏小型猪,克隆猪出生后根据表型再确认是否获得无毛猪。 结果： 1.构建携带西藏小型猪DKK1基因的慢病毒载体pERKDZG 成功克隆西藏小型猪DKK1基因,并成功构建慢病毒载体pEREZG,载体经酶切鉴定和测序,完全符合预期。 2.将目的基因导入西藏小型猪胚胎成纤维细胞 携带目的转基因ERKDZG的慢病毒感染PEFs后,发现仅有少量细胞发红色荧光,预示感染效率很低；然后对感染后的细胞进行扩大培养,流式细胞仪分选RFP阳性的细胞,最终获得纯的可用于体细胞核移植的经遗传修饰的PEFs,命名为PEF-DKK1。 RT-PCR检测显示,DKK1转基因在293T细胞中能够正常过表达。 3.K14启动子和转基因载体pERKDZG的体外功能验证 pERKDZG转染六种细胞后,发现成功转染pERKDZG的四种细胞中,皮肤细胞(B16)和非皮肤细胞(293T、PEFs和HNE1均有GFP和RFP表达；与RFP相对表达强度相比,皮肤细胞B16和293T上GFP表达相对较强,而其它细胞(PEFs和HNE1)上GFP表达相对较弱。总之,K14启动子和转基因载体pERKDZG的具有功能,可以进行下一步实验。 4.通过体细胞核移植技术制作DKKl转基因体细胞克隆西藏小型猪 成功进行卵母细胞的采集,体外成熟培养,核移植操作。重构胚胎体外培养7天,发育正常,囊胚率20%左右,重构胚全部表达RFP,部分表达GFP,预示目的基因已经转入重构胚并正常表达。共移植受体猪5头,其中两头怀孕足月产克隆猪8头,经PCR鉴定,其中3头为DKK1转基因克隆猪。DKK1转基因克隆猪表达RFP和GFP。 结论： 1.成功构建携带猪DKK1基因的慢病毒载体pERKDZG,并利用该慢病毒将西藏小型猪DKK1基因导入PEFs,进而流式分选获得纯的经遗传修饰的PEFs(命名为PEF-DKKl),其作为核供体细胞。 2.体外功能验证,K14启动子和转基因载体pERKDZG具有功能,可以进行后续实验。 3.通过体细胞核移植技术,成功获得3头携带西藏小型猪DKK1转基因的克隆猪,DKK1转基因克隆猪可正常表达RFP和GFP。 本研究的创新之处： 本研究利用我国独特的小型猪品系—西藏小型猪,将体细胞基因修饰和克隆技术相结合,培育具有我国自主知识产权的DKK1转基因体细胞克隆西藏小型猪,期望获得无毛西藏小型猪品系,以为皮肤移植、化妆品安全评价、紫外线照射、皮肤致癌、烧伤、冻伤、皮肤老化及抗老化等研究提供标准化的无毛实验小型猪。此外,也为研究人类毛发再生机制和脱发的基因治疗提供大型实验动物模型。
[Abstract]:Background and significance:
Because of its small size, ease of operation, and anatomy, physiological and biochemical characteristics and human * s characteristics, miniature pig has gradually become an important laboratory animal in the field of biomedicine research. The number of applications has been increasing year by year.
The * * Tibet miniature pig is one of the smaller pigs in the world. It comes from the Qinghai Tibet Plateau, the agricultural area and the farming and pastoral area * 2500-4300 meters above sea level. It is the only kind of pig that can adapt to the high altitude climate and grazing. The closed geographical environment has saved the very pure variety resources of the Tibet miniature pig. The experimental animal center of Southern Medical University In 2004, * Tibet miniature pig was introduced from Tibet to Guangzhou for laboratory animal research. At present, domestication and laboratory animal studies have been completed, and animal models, drug tests and transgenic cloning have been carried out. Immunology and genetics have revealed that the strain has its unique immune related indicators and genetic characteristics. With its unique shape, it is an excellent experimental miniature pig strain. *
* the skin system of miniature pig has a great similarity with human beings, so it is considered an ideal model for skin related research. * the skin of pigs and human beings is composed of three layers * epidermis, dermis and subcutaneous tissue. Pig skin is very similar to human skin in terms of anatomical structure, morphology, physiological and biochemical characteristics and pharmacology, including skin thickness (such as miniature pig skin). The skin is 70-140 micron thick, the human skin is 50-120 micron thick, and the skin is 10-20 micron thick, the skin morphology and proliferation kinetics, skin repair and regeneration, the humoral and metabolic changes of burn skin * and so on. Therefore, pigs are considered as a standard model for skin and plastic surgery, and also for skin transplantation, cosmetic safety evaluation, UV. Ideal experimental materials for line irradiation, skin carcinogenesis, burns, frostbite, skin aging and anti-aging. However, pig skin surface is always * * cumbersome before shaving or skin surgery.
The common hairless laboratory animals are nude mice, Yuyi hairless mice and hairless guinea pigs. Hairless guinea pigs have been widely used in skin related studies (such as hair growth agents, skin allergies, skin grafting and ultraviolet radiation). At present, the only hairless pig breed in the world is Yucatan * * (Yucatan) miniature pig, also called ink. There are many miniature pig strains in China: * * Five Fingers Group miniature pig, Guizhou miniature pig, Guangxi Bama miniature pig, Banna miniature pig and Tibet miniature pig * and so on. No hairless miniature pig breeding line has yet been produced, and biological medicine research is eager to create miniature pig strains based on China's breeding. New * hairless miniature pig.
The creation of * hairless miniature pig strains will relieve the complicated hair removal procedures and skin damage during the related experiments. The * hairless mini pigs can be widely used in skin transplantation, cosmetic safety evaluation, ultraviolet radiation, skin carcinogenesis, burns, frostbite, skin aging and anti-aging experiments, and also provide ideal for hair loss research. Large animal models are of great significance to the research of biomedicine in China.
A new experimental animal strain (e.g. white flower or hairless) is usually obtained by screening mutant phenotypes from naturally born animals and then inbreeding to obtain new strains with this phenotype. The previously mentioned Yuyi hairless mice and hairless guinea pigs were bred in this way. However, it is very difficult and unrealistic for large laboratory animals to acquire individuals with mutant phenotypes and then develop new strains by such methods.
It has been found that the hairless skin (i.e. hairless mice) of the transgenic mice with skin-specific overexpression of DKK1, which is regulated by the human skin-specific K14 promoter, is due to skin-specific overexpression of DKK1, which results in blocked hair development in mice, and the other aspects of the transgenic mice are in good condition. A large number of studies have shown that during the development of human and higher animals * such as mice and pigs, the coding sequence of the same gene (such as DKK1) is highly homologous, which determines that its function is often conserved among different species. * the coding sequence of pig DKK1 gene is highly homologous with human and mouse, which indicates the function of DKK1 gene. * humans, pigs and mice are also conserved (the same function). This suggests that transgenic porcine skin * * overexpression of porcine DKK1 gene may cause the development of DKK1 transgenic pig hair to be blocked and become hairless, so that no pig can be obtained.
In addition, a transgenic pig with specific skin specific expression of green fluorescent protein (GFP) gene (GFP expression regulated by human skin specific K14 promoter) was prepared by lentivirus vector method. The * * * confirmed that human K14 promoter could work normally in pigs, and the expression of GFP gene was restricted to pig skin, while no GFP expression was found in other tissues, indicating that human K14 started. It can control the specific expression of genetically modified * in pig skin tissue.
At present * there are mainly two ways to prepare transgenic pigs: somatic cell nuclear transfer and lentivirus vector *. The latter method is not allowed to be widely used in the preparation of transgenic pigs subject to the restriction of collection and quantity of fertilized eggs. * based on this, somatic cell nuclear transfer (somatic cell nuclear transfer) is used to prepare transgenic cloned pigs.
In summary, the aim of this study is to establish a transgenic porcine DKK1 transgenic pig * * * (DKK1 transgenic K14 gene promoter) which is controlled by human K14 promoter (transgenic DKK1 clone). The gene, which causes the development of * transgenic pigs to be blocked, becomes hairless, thus achieving the goal of creating new * hairless miniature pigs.
Based on the success of the Tibet miniature pig, the * * * is designed to create a new miniature hairless pig based on the other miniature pig * lines bred in China, so as to better meet the needs of multiple uses.
Method:
1. * construction of lentiviral vector pERKDZG carrying DKK1 gene of Tibet miniature pig
First, RNA was extracted from liver tissue of Tibet miniature pig, and then RT * * was used as cDNA. Primers were designed and amplified by PCR. The coding region of DKK1 gene of Tibet miniature pig (DKK1) was amplified. Then, 3 steps DNA cloning was performed by enzyme digestion and in-fusion cloning.
First, pDKK1 gene was inserted into the vector pK14-DKK1 to replace the mouse DKK1 fragment, and O was used to obtain the carrier pK14-pDKK1.
(2) The EF1a-RFP fragment was amplified from the vector pCDH-CMV-MCS-EF1a-RFP and inserted into the lentiviral vector pHAGE-fullEF1a-MCS-IzsGreen to obtain the vector pEREZG.
(3) K14-pDKK1 fragment was amplified from the vector pKl4-pDKK1 and inserted into the vector pEREZG to obtain the final lentiviral vector pERKDZG.
2. * introducing lentivirus into transgenic Tibet miniature pig embryo fibroblasts (PEFs).
PEFs were infected with lentiviral vector pERKDZG, and then infected with lentiviruses carrying the target gene ERKDZG. The introduction and expression of exogenous transgenic PEFs were monitored by RFP and GFP. The PEFs were cultured in vitro, and then the RFP positive cells were sorted by flow cytometry. Genetically modified PEFs, named PEF-DKK1.
Total RNA was extracted from 293 T cells transfected with pERKDZG during the production of lentivirus, and the expression of DKK1 gene was detected by RT-PCR.
In vitro functional verification of 3.K14 promoter and transgenic vector pERKDZG
PERKDZG was transfected into skin cells (* Tibet miniature pig skin fibroblast (PDFs), mouse skin melanoma cell line B-16] and non skin cells [i.e. PEFs, mouse embryonic fibroblast (MEFs), human nasopharyngeal carcinoma cell line HNE1 cells, 293T cells]. Then RFP and GFP were observed in the above six cells under inverted fluorescence microscope. Expression was used to verify the tissue specificity of K14 promoter and the function of transgenic vector pERKDZG.
4. producing DKK1 transgenic cell clone through somatic cell nuclear transfer * Tibet miniature pig.
* the porcine ovary was collected from slaughterhouses, and the oocytes were collected and matured in the laboratory. After 40 hours of maturation, hyaluronidase was removed, the oocytes were removed, and the PEF-DKK1 cells were injected into the zona pellucida of the enucleated oocytes. Then the electrofusion was activated, so that the reconstructed eggs began to develop. After reconstructing embryos in a short time, transplanted embryos into the oviduct of estrus sows at the early stage of 2-4 cell * * *, and producing transgenic cloned Tibetan miniature pigs depending on surrogate sows.
Result:
1. * construction of lentiviral vector pERKDZG carrying DKK1 gene of Tibetan miniature pig
The DKK1 gene of * Tibetan miniature pig was cloned successfully, and the lentiviral vector pEREZG was successfully constructed. The vector was identified and sequenced by restriction enzyme digestion.
2. * import the target gene into Tibetan miniature pig embryo fibroblasts.
After infected with PEFs by lentiviruses carrying ERKDZG, only a small number of cells showed red fluorescence, indicating low infection efficiency; then the infected cells were expanded and cultured, and the RFP positive cells were sorted by flow cytometry. Finally, pure genetically modified PEFs, named PEF-DKK1, were obtained for somatic cell nuclear transfer.
RT-PCR detection showed that DKK1 transgene could normally overexpress in 293T cells.
In vitro functional verification of 3.K14 promoter and transgenic vector pERKDZG
After transfection of pERKDZG into six kinds of cells, it was found that GFP and RFP were expressed in both skin cells (B16) and non-skin cells (293T, PEFs and HNE1); GFP expression was relatively strong in skin cells B16 and 293T, but weaker in other cells (PEFs and HNE1). And the function of transgenic vector pERKDZG can carry out the next experiment.
4. producing DKKl transgenic cell clone through somatic cell nuclear transfer * Tibet miniature pig.
Successful oocyte collection, in vitro maturation and nuclear transfer were performed. Reconstructed embryos were cultured in vitro for 7 days and grew normally. Blastocyst rate was about 20%. All reconstructed embryos expressed RFP and partially expressed GFP, indicating that the target gene had been transferred into the reconstructed embryos and expressed normally. 5 pigs were transplanted into the recipient * *, and two of them were pregnant and 8 of the cloned pigs were born in the full-term, and were identified by PCR. Of them, 3 of them were DKK1 * * transgenic pigs, and.DKK1 transgenic cloned pigs expressed RFP and GFP..
Conclusion:
1. * the lentiviral vector pERKDZG carrying pig DKK1 gene was successfully constructed, and the * DKK1 gene of Tibet mini pig was introduced into PEFs using the lentivirus. Then the purified genetically modified PEFs (named PEF-DKKl) was obtained by flow sorting and used as a nuclear donor cell.
2. in vitro functional verification, K14 promoter and transgenic vector pERKDZG have functions and can be followed up.
3. through somatic cell nuclear transfer technology, 3 cloned pigs carrying DKK1 transgenic * * * of Tibet miniature pigs were successfully obtained, and DKK1 transgenic cloned pigs could express RFP and GFP. normally.
The innovations of this research are:
In this * * *, we use the unique miniature pig strain Tibet miniature pig to combine somatic cell gene modification and cloning technology to cultivate DKK1 transgenic stem cell clone * Tibet miniature pig with independent intellectual property rights. We hope to obtain the hairless Tibet miniature pig strain, think skin grafting, cosmetic safety evaluation, ultraviolet radiation, skin. Studies on carcinogenesis, burns, frostbite, skin aging and anti aging * provide standardized hairless experimental miniature pigs. In addition, it provides a large experimental animal model for studying the mechanism of human hair regeneration and gene therapy for alopecia.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：S828;Q78
本文编号：2195957
[Abstract]:Background and significance:
Because of its small size, ease of operation, and anatomy, physiological and biochemical characteristics and human * s characteristics, miniature pig has gradually become an important laboratory animal in the field of biomedicine research. The number of applications has been increasing year by year.
The * * Tibet miniature pig is one of the smaller pigs in the world. It comes from the Qinghai Tibet Plateau, the agricultural area and the farming and pastoral area * 2500-4300 meters above sea level. It is the only kind of pig that can adapt to the high altitude climate and grazing. The closed geographical environment has saved the very pure variety resources of the Tibet miniature pig. The experimental animal center of Southern Medical University In 2004, * Tibet miniature pig was introduced from Tibet to Guangzhou for laboratory animal research. At present, domestication and laboratory animal studies have been completed, and animal models, drug tests and transgenic cloning have been carried out. Immunology and genetics have revealed that the strain has its unique immune related indicators and genetic characteristics. With its unique shape, it is an excellent experimental miniature pig strain. *
* the skin system of miniature pig has a great similarity with human beings, so it is considered an ideal model for skin related research. * the skin of pigs and human beings is composed of three layers * epidermis, dermis and subcutaneous tissue. Pig skin is very similar to human skin in terms of anatomical structure, morphology, physiological and biochemical characteristics and pharmacology, including skin thickness (such as miniature pig skin). The skin is 70-140 micron thick, the human skin is 50-120 micron thick, and the skin is 10-20 micron thick, the skin morphology and proliferation kinetics, skin repair and regeneration, the humoral and metabolic changes of burn skin * and so on. Therefore, pigs are considered as a standard model for skin and plastic surgery, and also for skin transplantation, cosmetic safety evaluation, UV. Ideal experimental materials for line irradiation, skin carcinogenesis, burns, frostbite, skin aging and anti-aging. However, pig skin surface is always * * cumbersome before shaving or skin surgery.
The common hairless laboratory animals are nude mice, Yuyi hairless mice and hairless guinea pigs. Hairless guinea pigs have been widely used in skin related studies (such as hair growth agents, skin allergies, skin grafting and ultraviolet radiation). At present, the only hairless pig breed in the world is Yucatan * * (Yucatan) miniature pig, also called ink. There are many miniature pig strains in China: * * Five Fingers Group miniature pig, Guizhou miniature pig, Guangxi Bama miniature pig, Banna miniature pig and Tibet miniature pig * and so on. No hairless miniature pig breeding line has yet been produced, and biological medicine research is eager to create miniature pig strains based on China's breeding. New * hairless miniature pig.
The creation of * hairless miniature pig strains will relieve the complicated hair removal procedures and skin damage during the related experiments. The * hairless mini pigs can be widely used in skin transplantation, cosmetic safety evaluation, ultraviolet radiation, skin carcinogenesis, burns, frostbite, skin aging and anti-aging experiments, and also provide ideal for hair loss research. Large animal models are of great significance to the research of biomedicine in China.
A new experimental animal strain (e.g. white flower or hairless) is usually obtained by screening mutant phenotypes from naturally born animals and then inbreeding to obtain new strains with this phenotype. The previously mentioned Yuyi hairless mice and hairless guinea pigs were bred in this way. However, it is very difficult and unrealistic for large laboratory animals to acquire individuals with mutant phenotypes and then develop new strains by such methods.
It has been found that the hairless skin (i.e. hairless mice) of the transgenic mice with skin-specific overexpression of DKK1, which is regulated by the human skin-specific K14 promoter, is due to skin-specific overexpression of DKK1, which results in blocked hair development in mice, and the other aspects of the transgenic mice are in good condition. A large number of studies have shown that during the development of human and higher animals * such as mice and pigs, the coding sequence of the same gene (such as DKK1) is highly homologous, which determines that its function is often conserved among different species. * the coding sequence of pig DKK1 gene is highly homologous with human and mouse, which indicates the function of DKK1 gene. * humans, pigs and mice are also conserved (the same function). This suggests that transgenic porcine skin * * overexpression of porcine DKK1 gene may cause the development of DKK1 transgenic pig hair to be blocked and become hairless, so that no pig can be obtained.
In addition, a transgenic pig with specific skin specific expression of green fluorescent protein (GFP) gene (GFP expression regulated by human skin specific K14 promoter) was prepared by lentivirus vector method. The * * * confirmed that human K14 promoter could work normally in pigs, and the expression of GFP gene was restricted to pig skin, while no GFP expression was found in other tissues, indicating that human K14 started. It can control the specific expression of genetically modified * in pig skin tissue.
At present * there are mainly two ways to prepare transgenic pigs: somatic cell nuclear transfer and lentivirus vector *. The latter method is not allowed to be widely used in the preparation of transgenic pigs subject to the restriction of collection and quantity of fertilized eggs. * based on this, somatic cell nuclear transfer (somatic cell nuclear transfer) is used to prepare transgenic cloned pigs.
In summary, the aim of this study is to establish a transgenic porcine DKK1 transgenic pig * * * (DKK1 transgenic K14 gene promoter) which is controlled by human K14 promoter (transgenic DKK1 clone). The gene, which causes the development of * transgenic pigs to be blocked, becomes hairless, thus achieving the goal of creating new * hairless miniature pigs.
Based on the success of the Tibet miniature pig, the * * * is designed to create a new miniature hairless pig based on the other miniature pig * lines bred in China, so as to better meet the needs of multiple uses.
Method:
1. * construction of lentiviral vector pERKDZG carrying DKK1 gene of Tibet miniature pig
First, RNA was extracted from liver tissue of Tibet miniature pig, and then RT * * was used as cDNA. Primers were designed and amplified by PCR. The coding region of DKK1 gene of Tibet miniature pig (DKK1) was amplified. Then, 3 steps DNA cloning was performed by enzyme digestion and in-fusion cloning.
First, pDKK1 gene was inserted into the vector pK14-DKK1 to replace the mouse DKK1 fragment, and O was used to obtain the carrier pK14-pDKK1.
(2) The EF1a-RFP fragment was amplified from the vector pCDH-CMV-MCS-EF1a-RFP and inserted into the lentiviral vector pHAGE-fullEF1a-MCS-IzsGreen to obtain the vector pEREZG.
(3) K14-pDKK1 fragment was amplified from the vector pKl4-pDKK1 and inserted into the vector pEREZG to obtain the final lentiviral vector pERKDZG.
2. * introducing lentivirus into transgenic Tibet miniature pig embryo fibroblasts (PEFs).
PEFs were infected with lentiviral vector pERKDZG, and then infected with lentiviruses carrying the target gene ERKDZG. The introduction and expression of exogenous transgenic PEFs were monitored by RFP and GFP. The PEFs were cultured in vitro, and then the RFP positive cells were sorted by flow cytometry. Genetically modified PEFs, named PEF-DKK1.
Total RNA was extracted from 293 T cells transfected with pERKDZG during the production of lentivirus, and the expression of DKK1 gene was detected by RT-PCR.
In vitro functional verification of 3.K14 promoter and transgenic vector pERKDZG
PERKDZG was transfected into skin cells (* Tibet miniature pig skin fibroblast (PDFs), mouse skin melanoma cell line B-16] and non skin cells [i.e. PEFs, mouse embryonic fibroblast (MEFs), human nasopharyngeal carcinoma cell line HNE1 cells, 293T cells]. Then RFP and GFP were observed in the above six cells under inverted fluorescence microscope. Expression was used to verify the tissue specificity of K14 promoter and the function of transgenic vector pERKDZG.
4. producing DKK1 transgenic cell clone through somatic cell nuclear transfer * Tibet miniature pig.
* the porcine ovary was collected from slaughterhouses, and the oocytes were collected and matured in the laboratory. After 40 hours of maturation, hyaluronidase was removed, the oocytes were removed, and the PEF-DKK1 cells were injected into the zona pellucida of the enucleated oocytes. Then the electrofusion was activated, so that the reconstructed eggs began to develop. After reconstructing embryos in a short time, transplanted embryos into the oviduct of estrus sows at the early stage of 2-4 cell * * *, and producing transgenic cloned Tibetan miniature pigs depending on surrogate sows.
Result:
1. * construction of lentiviral vector pERKDZG carrying DKK1 gene of Tibetan miniature pig
The DKK1 gene of * Tibetan miniature pig was cloned successfully, and the lentiviral vector pEREZG was successfully constructed. The vector was identified and sequenced by restriction enzyme digestion.
2. * import the target gene into Tibetan miniature pig embryo fibroblasts.
After infected with PEFs by lentiviruses carrying ERKDZG, only a small number of cells showed red fluorescence, indicating low infection efficiency; then the infected cells were expanded and cultured, and the RFP positive cells were sorted by flow cytometry. Finally, pure genetically modified PEFs, named PEF-DKK1, were obtained for somatic cell nuclear transfer.
RT-PCR detection showed that DKK1 transgene could normally overexpress in 293T cells.
In vitro functional verification of 3.K14 promoter and transgenic vector pERKDZG
After transfection of pERKDZG into six kinds of cells, it was found that GFP and RFP were expressed in both skin cells (B16) and non-skin cells (293T, PEFs and HNE1); GFP expression was relatively strong in skin cells B16 and 293T, but weaker in other cells (PEFs and HNE1). And the function of transgenic vector pERKDZG can carry out the next experiment.
4. producing DKKl transgenic cell clone through somatic cell nuclear transfer * Tibet miniature pig.
Successful oocyte collection, in vitro maturation and nuclear transfer were performed. Reconstructed embryos were cultured in vitro for 7 days and grew normally. Blastocyst rate was about 20%. All reconstructed embryos expressed RFP and partially expressed GFP, indicating that the target gene had been transferred into the reconstructed embryos and expressed normally. 5 pigs were transplanted into the recipient * *, and two of them were pregnant and 8 of the cloned pigs were born in the full-term, and were identified by PCR. Of them, 3 of them were DKK1 * * transgenic pigs, and.DKK1 transgenic cloned pigs expressed RFP and GFP..
Conclusion:
1. * the lentiviral vector pERKDZG carrying pig DKK1 gene was successfully constructed, and the * DKK1 gene of Tibet mini pig was introduced into PEFs using the lentivirus. Then the purified genetically modified PEFs (named PEF-DKKl) was obtained by flow sorting and used as a nuclear donor cell.
2. in vitro functional verification, K14 promoter and transgenic vector pERKDZG have functions and can be followed up.
3. through somatic cell nuclear transfer technology, 3 cloned pigs carrying DKK1 transgenic * * * of Tibet miniature pigs were successfully obtained, and DKK1 transgenic cloned pigs could express RFP and GFP. normally.
The innovations of this research are:
In this * * *, we use the unique miniature pig strain Tibet miniature pig to combine somatic cell gene modification and cloning technology to cultivate DKK1 transgenic stem cell clone * Tibet miniature pig with independent intellectual property rights. We hope to obtain the hairless Tibet miniature pig strain, think skin grafting, cosmetic safety evaluation, ultraviolet radiation, skin. Studies on carcinogenesis, burns, frostbite, skin aging and anti aging * provide standardized hairless experimental miniature pigs. In addition, it provides a large experimental animal model for studying the mechanism of human hair regeneration and gene therapy for alopecia.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：S828;Q78
【相似文献】
相关期刊论文 前10条
1 王爱德;;小型猪的利用价值及其品系繁育的进展[J];实验动物与比较医学;1990年02期
2 ;实验动物科学(Laboratory Animal Science)1986年 第36卷 第4期[J];实验动物科学与管理;1987年03期
3 ;中国实验用小型猪的培育、开发与应用[J];中国比较医学杂志;1991年01期
4 周忠信,王捷,罗葆明;小型猪肝硬化模型建立过程中肝脏功能及组织病理学的改变[J];上海实验动物科学;2002年02期
5 栗瑞福，李双良，，訾文敏;小型猪动脉压的测量[J];实验动物科学与管理;1996年01期
6 粟瑞福，乔伯英，黄丽洁;小型猪气管内插管技术介绍[J];实验动物科学与管理;1995年01期
7 粟瑞福，乔伯英，訾文敏;氯胺酮和戊巴比妥钠对于小型猪麻醉效果的观察[J];实验动物科学与管理;1995年01期
8 高青松;刘源;;实验用小型猪营养需要研究进展[J];中国比较医学杂志;2009年02期
9 孙国凤;;大阪大学组织关于开发实验动物小型猪的研究会[J];生物技术通报;1989年06期
10 郭亚芬，王爱德，兰干球，李柏;巴马小型猪染色体核型[J];上海实验动物科学;1994年01期
相关会议论文 前10条
1 洪丽玲;;小型猪作为一种动物模型在毒理学中的应用[A];2010年全国药物毒理学学术会议论文集[C];2010年
2 吕滨;;中华小型猪急性和慢性心肌缺血的综合影像学评价[A];中华医学会第十八次全国放射学学术会议论文汇编[C];2011年
3 袁进;顾为望;;小型猪作为人类疾病动物模型在生物医学研究中的应用前景[A];第九届中国实验动物科学年会（2010新疆）论文集[C];2010年
4 岳敏;范沛;巫玮;王玉珏;顾为望;;国内实验用小型猪循环系统疾病动物模型研究进展[A];第九届中国实验动物科学年会（2010新疆）论文集[C];2010年
5 马昆;张娈松;卢德章;范宏刚;姜岩;王洪斌;;XFM特异性颉颃剂对小型猪血常规的影响[A];中国畜牧兽医学会2010年学术年会——第二届中国兽医临床大会论文集(下册)[C];2010年
6 肖波;张小明;曾南林;蒲宇;唐伟;周立绥;左友波;庞君豪;;小型猪正常胰腺MRI表现及相关解剖[A];中华医学会第16次全国放射学学术大会论文汇编[C];2009年
7 鲍世民;张德福;高静华;马胜成;何新桥;朱庭玉;;小型猪专用S-99颗粒饲料研制及其评估[A];中国实验动物学会青年科技协会第二届学术研讨会论文汇编[C];2001年
8 詹纯利;徐本法;李建军;李权超;;小型猪剖腹产人工哺乳净化[A];中国实验动物学会第五届学术年会论文汇编[C];2000年
9 李兵;赵黎明;修清玉;沈俊;张慧;;CPAP对OSAS小型猪上气道结构和功能的影响[A];第八届全国生物力学学术会议论文集[C];2006年
10 鲍世民;马胜成;张德福;斋藤宗雄;谷冈功邦;何新桥;朱庭玉;;中国三品种小型猪血液生化正常值研究[A];中国动物科学研究——中国动物学会第十四届会员代表大会及中国动物学会65周年年会论文集[C];1999年
相关重要报纸文章 前10条
1 驻站记者 李云飞 通讯员 吕小飞;小型猪禽饲养带来大效益[N];赤峰日报;2007年
2 北京琉璃科兴试验动物养殖中心 孙国涛;小型猪的饲养和市场前景[N];中国特产报;2008年
3 常怀深;我国培育出首批体细胞克隆医用小型猪[N];农民日报;2007年
4 刘海峰;我国成功克隆出世界著名医用小型猪[N];新华每日电讯;2007年
5 记者 李立平;首批体细胞克隆医用小型猪培育成功[N];大众科技报;2007年
6 通讯员 吴京 记者 沈静;50年后 人体器官可能想换就换[N];医药导报;2007年
7 本版块稿件由山东省沂水县职教中心李连任提供;中小型猪场猪群保健实施方案[N];河南科技报;2004年
8 贾婧;五指山小型猪：人类疑难疾病的“替难者”[N];科技日报;2007年
9 樊丽;对中小型猪场经营失败的调查分析及对策探讨[N];中国畜牧报;2002年
10 记者 李志峰 实习生 姜阳;小香猪每月生活费上千元[N];重庆日报;2010年
相关博士学位论文 前10条
1 卢德章;小型猪特异性麻醉颉颃剂的研制及其催醒机理的研究[D];东北农业大学;2011年
2 胡魁;乳化异氟醚在小型猪全身麻醉中应用及其麻醉机理研究[D];东北农业大学;2013年
3 史若愚;分泌型蛋白DKK1在肝内胆管细胞癌侵袭转移中的作用及其机制研究[D];复旦大学;2012年
4 裴志勇;地黄低聚糖对同种异体脂肪组织来源干细胞移植治疗小型猪急性心肌梗死效应及机制的研究[D];中国人民解放军军医进修学院;2012年
5 陈云;小型猪实验动物化和糖尿病模型及中药“脾胃康”作用研究[D];广州中医药大学;2005年
6 裴志勇;地黄低聚糖对同种异体脂肪组织来源干细胞移植治疗小型猪急性心肌梗死效应及机制的研究[D];中国人民解放军医学院;2011年
7 谷新顺;冠脉内注射山莨菪碱对AMI-PCI后no-reflow现象逆转效应及no-reflow发生影响因素的研究[D];河北医科大学;2005年
8 于震;血栓性心肌缺血模型的建立及双参宁心胶囊的干预作用[D];中国中医科学院;2006年
9 魏静元;载脂蛋白CⅢ转基因小型猪高脂血症模型的建立与表型初步分析[D];吉林大学;2010年
10 单兆臣;水通道基因转导治疗小型猪腮腺放射损伤[D];首都医科大学;2004年
相关硕士学位论文 前10条
1 刘薇;DKK1转基因体细胞克隆西藏小型猪的制备[D];南方医科大学;2013年
2 张娈松;小型猪特异性麻醉颉颃剂对小型猪血流动力学的影响[D];东北农业大学;2011年
3 张念;中国特有小型猪内源性反转录病毒检测及变异特征研究[D];吉林大学;2013年
4 张宇沁;通过行为学观察研究小型猪经脉不通病理模型的方法学探索[D];中国中医科学院;2011年
5 马昆;小型猪特异性麻醉颉颃剂催醒效果综合监测[D];东北农业大学;2011年
6 赵斌;富血小板血浆对小型猪牙周膜细胞增殖和分化的影响[D];吉林大学;2011年
7 王欢;小型猪新型复合麻醉剂[D];南京农业大学;2011年
8 张晟;建立西藏小型猪诱导多能干细胞系初探[D];南方医科大学;2010年
9 杨同涛;小型猪乳化异氟醚麻醉的综合监测[D];东北农业大学;2013年
10 谭丽娟;阿替美唑及其复合制剂在小型猪催醒过程中的药代动力学研究[D];东北农业大学;2012年
本文编号：2195957
本文链接：https://www.wllwen.com/falvlunwen/zhishichanquanfa/2195957.html
