【摘要】：研究背景和意義： 由于體型小、便于操作,而且解剖、生理生化特性與人特別相近的特點,小型豬已逐漸成為生物醫(yī)藥研究領(lǐng)域重要的實驗動物,應(yīng)用數(shù)量逐年大幅遞增。 西藏小型豬是世界上體型較小的小型豬之一,來源于青藏高原、海拔2500-4300米的農(nóng)區(qū)和辦農(nóng)牧區(qū),是唯一能適應(yīng)高海拔氣候和以放牧為主的豬種,封閉的地理環(huán)境使西藏小型豬保存了非常純正的品種資源。南方醫(yī)科大學(xué)實驗動物中心于2004年將西藏小型豬從西藏引進(jìn)至廣州進(jìn)行實驗動物化研究,目前已完成風(fēng)土馴化及實驗動物化研究,并開展了相關(guān)動物模型、藥物試驗及轉(zhuǎn)基因克隆等研究。從免疫學(xué)、遺傳學(xué)等研究發(fā)現(xiàn),該品系具有其獨特的免疫相關(guān)指標(biāo)和遺傳特征,加上其獨特的外形,是一種優(yōu)良的實驗用小型豬品系。 小型豬的皮膚系統(tǒng)與人的有較大的相似性,故被認(rèn)為是皮膚相關(guān)研究的理想模型豬和人的皮膚均由表皮、真皮、皮下組織三層組成；豬的皮膚在解剖結(jié)構(gòu)、形態(tài)、生理生化特性與藥理學(xué)等方面與人的非常相似,包括皮膚厚薄(如小型豬皮膚70-140微米厚,人皮膚50-120微米厚,而大鼠皮膚10-20微米厚)、皮膚形態(tài)學(xué)和增生動力學(xué)、皮膚修復(fù)再生性、燒傷皮膚的體液和代謝變化規(guī)律等。因此,豬被認(rèn)為是皮膚和整形外科手術(shù)的標(biāo)準(zhǔn)模型,也是進(jìn)行皮膚移植、化妝品安全性評價、紫外線照射、皮膚致癌、燒傷、凍傷、皮膚的老化及抗老化等研究的理想實驗材料。但是,豬皮膚表面被毛,在對小型豬進(jìn)行皮膚實驗或外科手術(shù)之前,總是免不了繁瑣的剃毛程序。 常見的無毛小型實驗動物有裸鼠、豫醫(yī)無毛小鼠和無毛豚鼠,其中無毛豚鼠已被廣泛應(yīng)用于皮膚相關(guān)研究(如生發(fā)劑療效、皮膚過敏反應(yīng)、植皮治療、紫外線輻射反應(yīng)等)。目前,世界上僅有的一種無毛豬品系為尤卡坦(Yucatan)小型豬,也叫墨西哥無毛豬,廣泛應(yīng)用于皮膚研究。目前,我國已培育了許多小型豬品系：包括五指山小型豬、貴州小型豬、廣西巴馬小型豬、版納微型豬和西藏小型豬等,尚未培育出無毛小型豬品系,而生物醫(yī)學(xué)研究急盼基于中國培育的小型豬品系創(chuàng)制無毛小型豬新品系。 創(chuàng)制無毛小型豬品系,將免除相關(guān)實驗過程中繁雜的脫毛程序和由此造成的皮膚損傷,無毛小型豬可廣泛用于皮膚移植、化妝品安全性評價、紫外線照射、皮膚致癌、燒傷、凍傷、皮膚老化及抗老化等實驗,同時也為脫發(fā)研究提供了理想的大動物模型,對我國的生物醫(yī)藥研究有著重大意義。 通�？赏ㄟ^如下方法獲得一種新的實驗動物品系(如白花或無毛)：從自然出生的動物中篩選出具有突變表型的個體,然后進(jìn)行近交繁育,最終獲得具有此表型的新品系,前面提及的豫醫(yī)無毛小鼠和無毛豚鼠都是通過這種方法培育的。但是對于大型實驗動物來講,通過如上方法獲得具有突變表型的個體,并進(jìn)而培育成新的品系,是非常困難的和不太現(xiàn)實的。 已有研究發(fā)現(xiàn),皮膚特異性過表達(dá)小鼠DKK1轉(zhuǎn)基因(其表達(dá)受人皮膚特異性的K14啟動子調(diào)控)的轉(zhuǎn)基因小鼠皮膚無毛(即獲得了無毛小鼠),這是由于皮膚特異性過表達(dá)DKK1轉(zhuǎn)基因?qū)е滦∈竺l(fā)發(fā)育受阻,而該轉(zhuǎn)基因小鼠的其它方面情況正常。大量研究表明,在人和高等動物(如小鼠和豬等)的發(fā)育過程中,不同物種間的同一基因(如DKK1)的編碼序列高度同源,這決定其功能在不同物種間往往是保守的。豬DKK1基因的編碼序列與人和小鼠的高度同源,這些預(yù)示DKK1基因的功能在人、豬和小鼠等間亦是保守的(即功能相同),這提示在轉(zhuǎn)基因豬的皮膚特異性過表達(dá)豬DKK1轉(zhuǎn)基因,亦有可能導(dǎo)致該DKK1轉(zhuǎn)基因豬毛發(fā)發(fā)育受阻而變得無毛,從而獲得無毛豬。 此外,采用慢病毒載體法制備了皮膚特異性表達(dá)綠色熒光蛋白(GFP)基因(GFP表達(dá)受人皮膚特異性的K14啟動子調(diào)控)的轉(zhuǎn)基因豬,證實人K14啟動子在豬上能正常工作,且GFP基因的表達(dá)被限定在豬皮膚,而其它組織無GFP表達(dá),這預(yù)示人K14啟動子能控制其所調(diào)控的轉(zhuǎn)基因在豬皮膚組織特異性表達(dá)。 目前,制備轉(zhuǎn)基因豬的方法主要有兩種：體細(xì)胞核移植法和慢病毒載體法,其中后一種方法制備轉(zhuǎn)基因豬受制于受精卵采集和數(shù)量的限制,不能被普遍應(yīng)用�；诖�,本課題將采用體細(xì)胞核移植法制備轉(zhuǎn)基因克隆豬。 綜上,本課題擬借助轉(zhuǎn)基因體細(xì)胞核移植法,建立豬皮膚特異性過表達(dá)豬DKK1轉(zhuǎn)基因(豬DKK1轉(zhuǎn)基因的表達(dá)受人K14啟動子調(diào)控)的轉(zhuǎn)基因克隆小型豬(即制作DKK1轉(zhuǎn)基因體細(xì)胞克隆西藏小型豬),以實現(xiàn)在轉(zhuǎn)基因小型豬皮膚特異性過表達(dá)DKK1轉(zhuǎn)基因,進(jìn)而導(dǎo)致轉(zhuǎn)基因小型豬毛發(fā)發(fā)育受阻而變得無毛,從而實現(xiàn)創(chuàng)制無毛小型豬新品系的目標(biāo)。 本課題擬首先基于本中心培育的西藏小型豬創(chuàng)制無毛小型豬,如獲成功將進(jìn)一步基于中國培育的其它小型豬品系采用同樣的方法創(chuàng)制無毛小型豬新品系,以便更好滿足多方面的使用需求。 方法： 1.構(gòu)建攜帶西藏小型豬DKK1基因的慢病毒載體pERKDZG 首先從西藏小型豬肝臟組織提取RNA,逆轉(zhuǎn)錄為cDNA,設(shè)計引物并PCR擴(kuò)增西藏小型豬DKK1(簡寫為pDKK1)基因編碼區(qū)。然后通過酶切、in-fusion克隆的方法,進(jìn)行如下3步DNA克�。� ①將pDKK1基因插入載體pK14-DKK1,替換其中的鼠DKK1片段,以O(shè)獲得載體pK14-pDKK1; ②從載體pCDH-CMV-MCS-EF1a-RFP中擴(kuò)增EF1a-RFP片段,插入至慢病毒載體pHAGE-fullEF1a-MCS-IzsGreen,獲得載體pEREZG; ③從載體、pKl4-pDKK1中擴(kuò)增K14-pDKK1片段,插入載體pEREZG,獲得最終慢病毒載體pERKDZG。 2.借助慢病毒將目的轉(zhuǎn)基因?qū)胛鞑匦⌒拓i胚胎成纖維細(xì)胞(PEFs) 利用慢病毒載體pERKDZG包裝病毒,然后用攜帶目的轉(zhuǎn)基因ERKDZG的慢病毒感染PEFs,借助RFP和GFP監(jiān)測外源轉(zhuǎn)基因?qū)肱c表達(dá)情況。對病毒感染后的PEFs進(jìn)行擴(kuò)大培養(yǎng),然后借助流式細(xì)胞儀分選RFP陽性的細(xì)胞,最終獲得純的可用于體細(xì)胞核移植的經(jīng)遺傳修飾的PEFs,命名為PEF-DKK1。 從生產(chǎn)慢病毒時轉(zhuǎn)染了pERKDZG的293T細(xì)胞提取總RNA,進(jìn)行逆轉(zhuǎn)錄,RT-PCR檢測細(xì)胞中DKK1基因表達(dá)。 3.K14啟動子和轉(zhuǎn)基因載體pERKDZG的體外功能驗證 將pERKDZG分別轉(zhuǎn)染皮膚細(xì)胞[即西藏小型豬皮膚成纖維細(xì)胞(PDFs)、小鼠皮膚黑色素瘤細(xì)胞系B-16]和非皮膚細(xì)胞[即PEFs、小鼠胚胎成纖維細(xì)胞(MEFs)、人鼻咽癌細(xì)胞系HNE1細(xì)胞、293T細(xì)胞],然后在倒置熒光顯微鏡下觀察RFP和GFP在以上六種細(xì)胞中的表達(dá)情況,以驗證K14啟動子組織特異性和轉(zhuǎn)基因載體pERKDZG的功能性。 4.通過體細(xì)胞核移植技術(shù)制作DKK1轉(zhuǎn)基因體細(xì)胞克隆西藏小型豬 從屠宰場收集豬卵巢,實驗室收集卵母細(xì)胞,并進(jìn)行成熟培養(yǎng)。成熟培養(yǎng)40小時后,透明質(zhì)酸酶處理,脫去卵丘,顯微操作儀下進(jìn)行卵母細(xì)胞的去核,然后將PEF-DKK1細(xì)胞注射入去核卵母細(xì)胞透明帶下,然后電融合激活,使重構(gòu)卵開始發(fā)育,體外短暫培養(yǎng)重構(gòu)胚胎,將2-4細(xì)胞期的早期重構(gòu)胚胎移植入發(fā)情母豬的輸卵管,依賴代孕母豬生產(chǎn)轉(zhuǎn)基因克隆西藏小型豬,克隆豬出生后根據(jù)表型再確認(rèn)是否獲得無毛豬。 結(jié)果： 1.構(gòu)建攜帶西藏小型豬DKK1基因的慢病毒載體pERKDZG 成功克隆西藏小型豬DKK1基因,并成功構(gòu)建慢病毒載體pEREZG,載體經(jīng)酶切鑒定和測序,完全符合預(yù)期。 2.將目的基因?qū)胛鞑匦⌒拓i胚胎成纖維細(xì)胞 攜帶目的轉(zhuǎn)基因ERKDZG的慢病毒感染PEFs后,發(fā)現(xiàn)僅有少量細(xì)胞發(fā)紅色熒光,預(yù)示感染效率很低；然后對感染后的細(xì)胞進(jìn)行擴(kuò)大培養(yǎng),流式細(xì)胞儀分選RFP陽性的細(xì)胞,最終獲得純的可用于體細(xì)胞核移植的經(jīng)遺傳修飾的PEFs,命名為PEF-DKK1。 RT-PCR檢測顯示,DKK1轉(zhuǎn)基因在293T細(xì)胞中能夠正常過表達(dá)。 3.K14啟動子和轉(zhuǎn)基因載體pERKDZG的體外功能驗證 pERKDZG轉(zhuǎn)染六種細(xì)胞后,發(fā)現(xiàn)成功轉(zhuǎn)染pERKDZG的四種細(xì)胞中,皮膚細(xì)胞(B16)和非皮膚細(xì)胞(293T、PEFs和HNE1均有GFP和RFP表達(dá)；與RFP相對表達(dá)強(qiáng)度相比,皮膚細(xì)胞B16和293T上GFP表達(dá)相對較強(qiáng),而其它細(xì)胞(PEFs和HNE1)上GFP表達(dá)相對較弱�？傊�,K14啟動子和轉(zhuǎn)基因載體pERKDZG的具有功能,可以進(jìn)行下一步實驗。 4.通過體細(xì)胞核移植技術(shù)制作DKKl轉(zhuǎn)基因體細(xì)胞克隆西藏小型豬 成功進(jìn)行卵母細(xì)胞的采集,體外成熟培養(yǎng),核移植操作。重構(gòu)胚胎體外培養(yǎng)7天,發(fā)育正常,囊胚率20%左右,重構(gòu)胚全部表達(dá)RFP,部分表達(dá)GFP,預(yù)示目的基因已經(jīng)轉(zhuǎn)入重構(gòu)胚并正常表達(dá)。共移植受體豬5頭,其中兩頭懷孕足月產(chǎn)克隆豬8頭,經(jīng)PCR鑒定,其中3頭為DKK1轉(zhuǎn)基因克隆豬。DKK1轉(zhuǎn)基因克隆豬表達(dá)RFP和GFP。 結(jié)論： 1.成功構(gòu)建攜帶豬DKK1基因的慢病毒載體pERKDZG,并利用該慢病毒將西藏小型豬DKK1基因?qū)隤EFs,進(jìn)而流式分選獲得純的經(jīng)遺傳修飾的PEFs(命名為PEF-DKKl),其作為核供體細(xì)胞。 2.體外功能驗證,K14啟動子和轉(zhuǎn)基因載體pERKDZG具有功能,可以進(jìn)行后續(xù)實驗。 3.通過體細(xì)胞核移植技術(shù),成功獲得3頭攜帶西藏小型豬DKK1轉(zhuǎn)基因的克隆豬,DKK1轉(zhuǎn)基因克隆豬可正常表達(dá)RFP和GFP。 本研究的創(chuàng)新之處： 本研究利用我國獨特的小型豬品系—西藏小型豬,將體細(xì)胞基因修飾和克隆技術(shù)相結(jié)合,培育具有我國自主知識產(chǎn)權(quán)的DKK1轉(zhuǎn)基因體細(xì)胞克隆西藏小型豬,期望獲得無毛西藏小型豬品系,以為皮膚移植、化妝品安全評價、紫外線照射、皮膚致癌、燒傷、凍傷、皮膚老化及抗老化等研究提供標(biāo)準(zhǔn)化的無毛實驗小型豬。此外,也為研究人類毛發(fā)再生機(jī)制和脫發(fā)的基因治療提供大型實驗動物模型。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：S828;Q78

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王愛德;;小型豬的利用價值及其品系繁育的進(jìn)展[J];實驗動物與比較醫(yī)學(xué);1990年02期

2 ;實驗動物科學(xué)(Laboratory Animal Science)1986年 第36卷 第4期[J];實驗動物科學(xué)與管理;1987年03期

3 ;中國實驗用小型豬的培育、開發(fā)與應(yīng)用[J];中國比較醫(yī)學(xué)雜志;1991年01期

4 周忠信,王捷,羅葆明;小型豬肝硬化模型建立過程中肝臟功能及組織病理學(xué)的改變[J];上海實驗動物科學(xué);2002年02期

5 栗瑞福，李雙良，，訾文敏;小型豬動脈壓的測量[J];實驗動物科學(xué)與管理;1996年01期

6 粟瑞福，喬伯英，黃麗潔;小型豬氣管內(nèi)插管技術(shù)介紹[J];實驗動物科學(xué)與管理;1995年01期

7 粟瑞福，喬伯英，訾文敏;氯胺酮和戊巴比妥鈉對于小型豬麻醉效果的觀察[J];實驗動物科學(xué)與管理;1995年01期

8 高青松;劉源;;實驗用小型豬營養(yǎng)需要研究進(jìn)展[J];中國比較醫(yī)學(xué)雜志;2009年02期

9 孫國鳳;;大阪大學(xué)組織關(guān)于開發(fā)實驗動物小型豬的研究會[J];生物技術(shù)通報;1989年06期

10 郭亞芬，王愛德，蘭干球，李柏;巴馬小型豬染色體核型[J];上海實驗動物科學(xué);1994年01期

相關(guān)會議論文 前10條

1 洪麗玲;;小型豬作為一種動物模型在毒理學(xué)中的應(yīng)用[A];2010年全國藥物毒理學(xué)學(xué)術(shù)會議論文集[C];2010年

2 呂濱;;中華小型豬急性和慢性心肌缺血的綜合影像學(xué)評價[A];中華醫(yī)學(xué)會第十八次全國放射學(xué)學(xué)術(shù)會議論文匯編[C];2011年

3 袁進(jìn);顧為望;;小型豬作為人類疾病動物模型在生物醫(yī)學(xué)研究中的應(yīng)用前景[A];第九屆中國實驗動物科學(xué)年會（2010新疆）論文集[C];2010年

4 岳敏;范沛;巫瑋;王玉玨;顧為望;;國內(nèi)實驗用小型豬循環(huán)系統(tǒng)疾病動物模型研究進(jìn)展[A];第九屆中國實驗動物科學(xué)年會（2010新疆）論文集[C];2010年

5 馬昆;張孌松;盧德章;范宏剛;姜巖;王洪斌;;XFM特異性頡頏劑對小型豬血常規(guī)的影響[A];中國畜牧獸醫(yī)學(xué)會2010年學(xué)術(shù)年會——第二屆中國獸醫(yī)臨床大會論文集(下冊)[C];2010年

6 肖波;張小明;曾南林;蒲宇;唐偉;周立綏;左友波;龐君豪;;小型豬正常胰腺MRI表現(xiàn)及相關(guān)解剖[A];中華醫(yī)學(xué)會第16次全國放射學(xué)學(xué)術(shù)大會論文匯編[C];2009年

7 鮑世民;張德福;高靜華;馬勝成;何新橋;朱庭玉;;小型豬專用S-99顆粒飼料研制及其評估[A];中國實驗動物學(xué)會青年科技協(xié)會第二屆學(xué)術(shù)研討會論文匯編[C];2001年

8 詹純利;徐本法;李建軍;李權(quán)超;;小型豬剖腹產(chǎn)人工哺乳凈化[A];中國實驗動物學(xué)會第五屆學(xué)術(shù)年會論文匯編[C];2000年

9 李兵;趙黎明;修清玉;沈俊;張慧;;CPAP對OSAS小型豬上氣道結(jié)構(gòu)和功能的影響[A];第八屆全國生物力學(xué)學(xué)術(shù)會議論文集[C];2006年

10 鮑世民;馬勝成;張德福;齋藤宗雄;谷岡功邦;何新橋;朱庭玉;;中國三品種小型豬血液生化正常值研究[A];中國動物科學(xué)研究——中國動物學(xué)會第十四屆會員代表大會及中國動物學(xué)會65周年年會論文集[C];1999年

相關(guān)重要報紙文章 前10條

1 駐站記者 李云飛 通訊員 呂小飛;小型豬禽飼養(yǎng)帶來大效益[N];赤峰日報;2007年

2 北京琉璃科興試驗動物養(yǎng)殖中心 孫國濤;小型豬的飼養(yǎng)和市場前景[N];中國特產(chǎn)報;2008年

3 常懷深;我國培育出首批體細(xì)胞克隆醫(yī)用小型豬[N];農(nóng)民日報;2007年

4 劉海峰;我國成功克隆出世界著名醫(yī)用小型豬[N];新華每日電訊;2007年

5 記者 李立平;首批體細(xì)胞克隆醫(yī)用小型豬培育成功[N];大眾科技報;2007年

6 通訊員 吳京 記者 沈靜;50年后 人體器官可能想換就換[N];醫(yī)藥導(dǎo)報;2007年

7 本版塊稿件由山東省沂水縣職教中心李連任提供;中小型豬場豬群保健實施方案[N];河南科技報;2004年

8 賈婧;五指山小型豬：人類疑難疾病的“替難者”[N];科技日報;2007年

9 樊麗;對中小型豬場經(jīng)營失敗的調(diào)查分析及對策探討[N];中國畜牧報;2002年

10 記者 李志峰　實習(xí)生 姜陽;小香豬每月生活費上千元[N];重慶日報;2010年

相關(guān)博士學(xué)位論文 前10條

1 盧德章;小型豬特異性麻醉頡頏劑的研制及其催醒機(jī)理的研究[D];東北農(nóng)業(yè)大學(xué);2011年

2 胡魁;乳化異氟醚在小型豬全身麻醉中應(yīng)用及其麻醉機(jī)理研究[D];東北農(nóng)業(yè)大學(xué);2013年

3 史若愚;分泌型蛋白DKK1在肝內(nèi)膽管細(xì)胞癌侵襲轉(zhuǎn)移中的作用及其機(jī)制研究[D];復(fù)旦大學(xué);2012年

4 裴志勇;地黃低聚糖對同種異體脂肪組織來源干細(xì)胞移植治療小型豬急性心肌梗死效應(yīng)及機(jī)制的研究[D];中國人民解放軍軍醫(yī)進(jìn)修學(xué)院;2012年

5 陳云;小型豬實驗動物化和糖尿病模型及中藥“脾胃康”作用研究[D];廣州中醫(yī)藥大學(xué);2005年

6 裴志勇;地黃低聚糖對同種異體脂肪組織來源干細(xì)胞移植治療小型豬急性心肌梗死效應(yīng)及機(jī)制的研究[D];中國人民解放軍醫(yī)學(xué)院;2011年

7 谷新順;冠脈內(nèi)注射山莨菪堿對AMI-PCI后no-reflow現(xiàn)象逆轉(zhuǎn)效應(yīng)及no-reflow發(fā)生影響因素的研究[D];河北醫(yī)科大學(xué);2005年

8 于震;血栓性心肌缺血模型的建立及雙參寧心膠囊的干預(yù)作用[D];中國中醫(yī)科學(xué)院;2006年

9 魏靜元;載脂蛋白CⅢ轉(zhuǎn)基因小型豬高脂血癥模型的建立與表型初步分析[D];吉林大學(xué);2010年

10 單兆臣;水通道基因轉(zhuǎn)導(dǎo)治療小型豬腮腺放射損傷[D];首都醫(yī)科大學(xué);2004年

相關(guān)碩士學(xué)位論文 前10條

1 劉薇;DKK1轉(zhuǎn)基因體細(xì)胞克隆西藏小型豬的制備[D];南方醫(yī)科大學(xué);2013年

2 張孌松;小型豬特異性麻醉頡頏劑對小型豬血流動力學(xué)的影響[D];東北農(nóng)業(yè)大學(xué);2011年

3 張念;中國特有小型豬內(nèi)源性反轉(zhuǎn)錄病毒檢測及變異特征研究[D];吉林大學(xué);2013年

4 張宇沁;通過行為學(xué)觀察研究小型豬經(jīng)脈不通病理模型的方法學(xué)探索[D];中國中醫(yī)科學(xué)院;2011年

5 馬昆;小型豬特異性麻醉頡頏劑催醒效果綜合監(jiān)測[D];東北農(nóng)業(yè)大學(xué);2011年

6 趙斌;富血小板血漿對小型豬牙周膜細(xì)胞增殖和分化的影響[D];吉林大學(xué);2011年

7 王歡;小型豬新型復(fù)合麻醉劑[D];南京農(nóng)業(yè)大學(xué);2011年

8 張晟;建立西藏小型豬誘導(dǎo)多能干細(xì)胞系初探[D];南方醫(yī)科大學(xué);2010年

9 楊同濤;小型豬乳化異氟醚麻醉的綜合監(jiān)測[D];東北農(nóng)業(yè)大學(xué);2013年

10 譚麗娟;阿替美唑及其復(fù)合制劑在小型豬催醒過程中的藥代動力學(xué)研究[D];東北農(nóng)業(yè)大學(xué);2012年

本文編號：2195957