山羊β-casein位点打靶载体在乳腺上皮细胞中的表达研究

以本地山羊基因组DNA为模板,通过长链PCR扩增出山羊β-casein上游包括启动子,外显子1及部分外显子2的6.1kb的调控序列及下游3.3kb的序列,将来自质粒pCDNA3的neo基因以及来自质粒pNEOZTK-2的tk基因,经克隆重组后构建了本地山羊乳腺特异性定点打靶载体,并在其中克隆人乳铁蛋白mini基因,采用脂质体法转染小鼠乳腺上皮癌化细胞系C127,以进行打靶载体的表达功能检测,双夹心ELISA测得诱导液中乳铁蛋白表达量为0.2μg/mL,Western-blot显示重组蛋白分子量比标准品略小,约为76kD,结果说明本载体能够指导外源基因在动物乳腺细胞内正确表达。     

Targeting the exogenous htPAm gene on goat somatic cell beta-casein locus for transgenic goat production

Combining gene targeting of animal somatic cells with nuclear transfer technique has provided a powerful method to produce transgenic animal mammary gland bioreactor. The objective of this study is to make an efficient and reproducible gene targeting in goat fetal fibroblasts by inserting the exogenous htPAm cDNA into the beta-casein locus with liposomes or electroporation so that htPAm protein might be produced in gene-targeted goat mammary gland. By gene-targeting technique, the exogenous htPAm gene was inserted to milk goat beta-casein gene sequences. Fetal fibroblasts were isolated from Day 35 fetuses of Guanzhong milk goats, and transfected with linear gene-targeting vector pGBC4htPAm using Lipefectamin-2000 and electoporation, respectively. Forty-eight gene-targeted cell colonies with homologous recombination were obtained, and three cell colonies were verified by DNA sequence analysis within the homologous recombination region. Using gene-targeted cell lines as donor cells for nuclear transfer, a total of 600 reconstructed embryos had been obtained, and 146 developed cloned embryos were transferred to 16 recipient goats, and finally three goats showed pregnancy at Day 90.     

ht-Pam基因在山羊β-酪蛋白基因座定位整合的研究

利用体细胞基因打靶与核移植技术制备动物乳腺生物反应器是当今转基因定位整合表达的一种新技术。分别克隆山羊的β-酪蛋白基因5′调控区的6.3kb片段,外显子7、外显子8和9三个基因片段,并与克隆的人tPA突变体cDNA一起构建了含有neo和tk正负筛选标记基因的β-酪蛋白基因打靶载体P^GBC4tPA,并验证了neo基因、tk基因以及Cre-LoxP系统的有效性。将线性化的P^GBC4tPA通过电转染整合到山羊胎儿成纤维细胞基因组中,利用G418和GANC进行抗性细胞克隆的药物筛选,初步获得抗性细胞克隆244个,PCR检测后获得阳性细胞克隆31个,其中初步验证2个细胞克隆转植基因整合位点重组后的基因序列正确,并且该细胞克隆能够有效扩增。这为下一步基因打靶体细胞核移植制备山羊乳腺生物反应器奠定了基础。     

Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene

Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropri-ate post-translational modifications.The nuclear transfer of transgenic somatic cells is a powerful method to pro-duce mammary gland bioreactors.We established an effi-cient gene transfer and nuclear transfer approach in goat somatic cells.Gene targeting vector pGBC2LF was con-structed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene.Goat fetal fibroblasts were transfected with lin-earized pGBC2LF and 14 cell lines were positive accord-ing to PCR and Southern blot.The transgenic cells were used as donor cells of nuclear transfer and some of recon-structed embryos could develop into blastocyst in vitro.     

人乳铁蛋白cDNA 基因乳腺表达载体的构建与鉴定

为了构建人乳铁蛋白基因 (hLF) 的乳腺表达载体并验证其在乳腺细胞中的表达情况,本载体以山羊β-casein基因上游包括启动子、外显子1、内含子1、部分外显子2作为5′端调控序列,下游包括部分外显子7、内含子7、外显子8、内含子8、外显子9及3′部分基因组片段作为3′端调控序列,长度分别为6.2 kb和7.1 kb,将hLF基因 (目的基因) 和Neo基因 (筛选标记) 分别插入到5′端调控序列和3′端调控序列的下游,构建成pBC1-hLF-Neo载体,其全长为25.348 kb。为了检测该载体的生物学     

整合人lactoferrin基因的山羊体细胞支持核移植克隆胚的体外发育

克隆了人lactoferrin基因和山羊[[beta]]-casein基因5′端调控区, 构建了人lactoferrin的乳腺表达载体, 并将该载体利用脂质体介导转染了奶山羊胎儿成纤维细胞, 获得了稳定整合人lactoferrin基因的转基因体细胞克隆17个, 其中PCR和Southern Blot检测阳性的细胞克隆14个, 阳性率82.4%。以转基因体细胞为供体细胞进行了核移植, 获得了能够体外发育的山羊转基因克隆胚胎, 体内成熟卵母细胞来源的核移植囊胚率为64.8%, 体外成熟卵母细胞来源的核移植囊胚率为51.7%, 证明了山羊转基因体细胞能够支持克隆胚的进一步发育。     

牛乳铁蛋白肽转基因小鼠的构建

目的:探讨牛乳铁蛋白肽在转基因鼠乳汁中的表达及其抑菌活性。方法:将实验室构建并保存的包含山羊β-酪蛋白基因启动子和牛乳铁蛋白肽基因的乳腺特异表达载体PI-bcp-LfcinB,用Xho I和Nru I双酶切,得到含有全部表达盒的显微注射DNA片段,采用常规显微注射技术获得转基因小鼠。并通过对泌乳期转基因雌鼠乳腺组织的RT-PCR检测,确定了牛乳铁蛋白肽在mRNA水平的表达,同时利用琼脂板扩散法检测了转基因鼠乳汁中表达产物的抑菌活性。结果:获得了牛乳铁蛋白肽转基因小鼠,且转基因小鼠乳汁中能够表达具有抑菌活性的牛乳铁蛋白肽。结论:通过转基因动物乳腺可以获得具有生物活性的牛乳铁蛋白肽,为进一步研究抗菌肽转基因牛、培育抗乳房炎奶牛新品种以及通过建立转基因动物生物反应器进行抗菌肽的大量生产奠定了基础。     

转基因动物乳腺暂时性表达系统的建立

以乳清酸蛋白(WAP)基因5′区为调控序列,人基因组G-CSF基因为目的片段,同时将WAP基因第一内含子插入G-CSF基因5′端,构建成转基因动物乳腺表达载体。将其直接注射到小鼠乳腺,在泌乳期表达出人G-CSF。表明乳腺直接注射质粒的方法可以做为暂时性的一种表达系统,同时也表明内含子对表达有一定的作用。     

Production GH transgenic goat improving mammogenesis by somatic cell nuclear transfer

Growth hormone is a positive regulator of mammary gland development. Dairy animals that are administered growth hormone display enhanced lactation performance, a desirable agricultural trait. The objective of the current research was to generate an improved milk production phenotype in a large animal model using over-expressed GH in the mammary gland to promote mammogenesis. To this end, we constructed a mammary gland-specific expression vector, pcGH, and demonstrated effective GH expression in goat mammary epithelial cells in vitro by ELISA. Then, to produce transgenic offspring that were capable of stable GH expression in vivo, the linearized pcGH vector was electroporated into goat fetal fibroblasts. Cell colonies that were positive for GH were used as donors for nuclear transfer to enucleated oocytes. A total of 253 morulae or blastocytes developed from the reconstructed embryos were transferred to 56 recipients, resulting in 24 pregnancies at day 35. Finally, six transgenic goats were born. PCR detection confirmed the success of the cloning procedure. To observe the mammogenesis of dairy goats, the GH transgenic goats were mated with a completely healthy buck. In the later pregnancy period, the mammary gland of the GH transgenic goats were extensive than non-transgenic goats. These experiments indicated that the pcGH vector was incorporated into the transgenic goats and affected mammogenesis, which laid a solid foundation for elucidating the impact of GH on mammogenesis and lactation performance.     

Differential regulation of rat beta-casein-chloramphenicol acetyltransferase fusion gene expression in transgenic mice.

Previous studies in our laboratory have demonstrated the mammary-specific expression of the entire rat beta-casein gene with 3.5 kilobases (kb) of 5' and 3.0 kb of 3' DNA in transgenic mice (Lee et al., Nucleic Acids Res. 16:1027-1041, 1988). In an attempt to localize sequences that dictate this specificity, lines of transgenic mice carrying two different rat beta-casein promoter-bacterial chloramphenicol acetyltransferase (cat) fusion genes have been established. Twenty and eight lines of transgenic mice carrying two fusion genes containing either 2.3 or 0.5 kb, respectively, of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A were identified, most of which transmitted the transgenes to their offspring in a Mendelian pattern. CAT activity was detected predominantly in the lactating mammary gland of female transgenic mice but not in the male mammary fat pad. A several-hundred-fold variation in the level of cat expression was observed in the mammary gland of different lines of mice, presumably due to the site of integration of the transgenes. CAT activity was increased in the mammary gland during development from virgin to midpregnancy and lactation. Unexpectedly, the casein-cat transgenes were also expressed in the thymus of different lines of both male and female mice, in some cases at levels equivalent to those observed in the mammary gland, and in contrast to the mammary gland, CAT activity was decreased during pregnancy and lactation in the thymus. Thus, 0.5 kb of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A are sufficient to target bacterial cat gene expression to the mammary gland of lactating mice.     

表达人尿激酶原突变体的重组山羊乳腺特异性表达慢病毒载体的构建

目的：构建山羊乳腺特异性表达尿激酶原突变体的重组慢病毒载体,证明其表达的有效性。方法：将劳氏肉瘤病毒增强子／启动子、复制缺陷型人免疫缺陷病毒（HIV-1）的5′端长重复序列（LTR）、HIV-1ψ包装信号、HIVRev反应元件、山羊β-酪蛋白调控序列、尿激酶原M13cDNA、AU3／3′LTR、牛生长激素（BGH）基因poly（A）依次连接,构建乳腺特异性表达的慢病毒载体,通过体外转染人乳腺癌细胞系MCF-7、中国仓鼠卵巢细胞及泌乳山羊乳腺注射证明其表达有效性。结果：酶切鉴定证实山羊乳腺特异性表达载体构建正确;将该载体转染细胞,采用溶圈法和Western印迹检测证实了其表达的有效性;慢病毒载体注射到泌乳山羊的乳腺,在乳汁中也检测到了尿激酶原的表达。结论：为在转基因动物乳腺中表达尿激酶原突变体奠定了基础。     

乳腺特异高表达人促红细胞生成素转基因奶山羊的制备

目的制备乳腺特异性高表达人促红细胞生成素（hEPO）转基因奶山羊。方法采用牛β-乳球蛋白基因（BLG）调控元件和hEPO全长编码序列基因组DNA构建真核表达载体,应用受精卵原核注射的方法制备hEPO转基因山羊。结果在原核注射获得的188头羔羊中,经Southern blot法检测有4头羊含有hEPO基因,其中3头为母羊,1头公羊于出生后20d死亡;3头转基因母羊hEPO基因的拷贝数分别为1、10、2;Western blot检测结果显示转基因羊乳中的hEPO分子质量为32kDa;MTT法检测结果表明,在泌乳10d的3只转基因羊乳汁中,每毫升乳汁中hEPO活性分别达到1.17×10^2IU、1.90×10^4IU、1.91×10^4IU。结论牛BLG能够调控hEPO基因在山羊乳腺中高表达,为实现其他药用蛋白在山羊乳腺中表达奠定了基础。     

山羊PTHrP基因干扰重组腺病毒的制备与鉴定

甲状旁腺激素相关蛋白 (Parathyroid hormone related protein,PTHrP) 具有广泛生物学功能,对调控钙代谢具有重要作用。采用RNA干扰和重组腺病毒技术,对山羊乳腺上皮细胞中PTHrP基因表达进行沉默,为进一步研究该基因在乳腺上皮细胞中的功能奠定基础。采用BLOCK-iT shRNA腺病毒干扰系统,将设计好的寡聚shRNA-322/357经退火复性后插入穿梭质粒pENTR/CMV-GFP/U6中。经Western blotting检测干扰效率后,选择pENTR/CMV-GFP/U6-322与病毒骨架质粒pAd/PL-DEST进行同源重组,成功获得重组干扰腺病毒载体pAD/PL-DEST/CMV-GFP/U6-322,并转染HEK-293细胞,通过包装、扩增后产生干扰重组腺病毒AD-PTHrP-322,TCID50法测定其滴度为2.0×109 PFU/mL。用AD-PTHrP-322感染山羊原代乳腺上皮细胞(MOI=200),经实时定量PCR及Western blotting检测表明在病毒感染24 h、48 h和72 h后,山羊乳腺上皮细胞中PTHrP mRNA表达量分别下调了29.2％、68.1％和82.6％ (P＜0.05),其蛋白表达水平也明显下调,干扰效果明显。     

Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene

Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropriate post-translational modifications. The nuclear transfer of transgenic somatic cells is a powerful method to produce mammary gland bioreactors. We established an efficient gene transfer and nuclear transfer approach in goat somatic cells. Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer and some of reconstructed embryos could develop into blastocyst in vitro. __________ Translated from Hereditas (Beijing), 2006, 28(12): 1513–1519 [译自: 遗传]     

tPA/gGH双基因转染山羊乳腺上皮细胞表达分析

人组织纤溶酶原激活剂(tissue-type plasminogen activator,tPA)是一种被广泛应用于临床的溶栓药物。双基因共整合入生物体内能够产生协同作用,从而提高目的基因的表达水平。但是目前,利用gGH基因与tPA基因共整合以期提高tPA表达水平的相关研究较少。为筛选获得tPA高表达的tPA/gGH双基因整合的单克隆转基因山羊乳腺上皮细胞株,本研究以β-casein基因作为调控序列,构建乳腺特异性表达载体PCL25/gGH,并通过电转染将tPA和gGH双基因共转染山羊乳腺上皮细胞;通过G418筛选获得抗性细胞株,经PCR检测获得转基因单克隆细胞株;利用催乳素诱导tPA表达,收集48 h后细胞诱导液进行ELISA()和Western blotting检测并分析其tPA表达水平。结果表明,共获得142株抗性单克隆细胞,其中有53株tPA单基因整合细胞株,34株tPA/gGH双基因整合细胞株,双基因整合率达23.9%(34/142)。共检测出29株细胞能够表达tPA,其中单基因表达细胞为12株,表达率为22.6%(12/53);双基因表达细胞为17株,表达率为50.0%(17/34);且单基因细胞表达tPA含量为7.5~52.0μg/mL,而双基因细胞表达tPA含量为40~360μg/mL,明显高于单基因表达水平。本研究通过电转染的方式成功获得了tPA/gGH双基因整合的单克隆山羊乳腺上皮细胞株,并证明双基因整合的细胞株表达tPA水平明显提高,为后期制备高表达转基因山羊奠定了基础。     

牛乳腺核基质附着区的克隆及其功能研究

从奶牛的全血中提取基因组DNA,参照GenBank已有序列,通过Primer5.0和Vector 7.0辅助设计一对引物,克隆了牛乳腺核基质附着区(BMRs)。利用生物学软件对其进行初步分析后,TA克隆于pMD－18T vector上。上下游引物5′端分别引入Kpn2Ⅰ和XhoⅠ酶切位点,将所得的BMRs克隆到pEGFP-C1的报告基因下游,构建成表达载体BE,脂质体法转染牛耳成纤维细胞。以转染pEGFP-C1的牛耳成纤维细胞作为对照,发现所克隆的BMRs功能明显,在消除位置效应、增强报告基因表达方面具有一定的作用。     

奶山羊乳腺上皮细胞的分离、培养及鉴定

应用组织块培养法高密度培养、连续传代法建立西农萨能奶山羊乳腺上皮细胞体外培养体系,通过生长曲线绘制、核型分析、免疫荧光染色 (角蛋白、上皮膜抗原、波形蛋白、β-酪蛋白)、油红染色及β-酪蛋白基因的RT-PCR分析进行培养细胞鉴定。实验结果表明细胞生长曲线为典型的S型,染色体数目众数为60,细胞角蛋白、上皮膜抗原、波形蛋白、β-酪蛋白表达均呈阳性,油红染色后可见细胞质内的脂滴,且细胞表达酪蛋白mRNA。说明运用本方法培养的细胞为正常的乳腺上皮细胞,并具有一定的泌乳功能。     

北川山羊PPARD基因克隆及组织表达分析

本试验从北川山羊的乳腺组织得到PPARD基因序列,并对其特有的生物学特点进行分析,同时将其在不同组织中所表达的情况进行阐述。利用RT-PCR的技术克隆获得北川山羊PPARD基因的序列,采用实时荧光定量PCR检测PPARD基因在北川山羊不同组织和泌乳期的表达丰度。结果表明,所得的北川山羊PPARD基因序列的CDs区有1 326 bp(登录号:XM018039044),由441个氨基酸编码,是不稳定的亲水性蛋白,不存在信号肽序列和跨膜结构。PPARD基因在北川山羊的表达水平最高的是脂肪组织,极显著高于其他组织(p<0.01),在乳腺组织和胃中也有较高的表达。其在泌乳早期的表达水平极显著高于空怀期和干奶期,这为进一步研究PPARD基因在北川山羊乳腺脂代谢过程所起的作用提供了理论基础。     

In vitro evaluation of a mammary gland specific expression vector encoding recombinant human lysozyme for development of transgenic dairy goat embryos

A vector expressing human lysozyme (pBC1-hLYZ-GFP-Neo) was evaluated for gene and protein expression following liposome-mediated transformation of C-127 mouse mammary cancer cells. Cultures of G418-resistant clones were harvested 24-72 h after induction with prolactin, insulin and hydrocortisone. Target gene expression was analyzed by RT-PCR and Western blot and recombinant human lysozyme (rhLYZ) bacteriostatic activity was also evaluated. The hLYZ gene was correctly transcribed and translated in C-127 cells and hLYZ inhibited gram-positive bacterial growth, indicating the potential of this expression vector for development of a mammary gland bioreactor in goats. Guanzhong dairy goat skin fibroblasts transfected with pBC1-hLYZ-GFP-Neo were used to construct a goat embryo transgenically expressing rhLYZ by somatic nuclear transplantation with a blastocyst rate of 9.0 ± 2.8 %. These data establish the basis for cultivation of mastitis-resistant hLYZ transgenic goats.     

The onset of mammary extraction of plasma triglycerides and circulating levels of progesterone during lactogenesis in the cow and goat

In untreated cows and goats, the onset of mammary extraction of plasma triglyceride was sometimes detected several hours before parturition, prior to removal of secretion from the mammary glands by offspring and when circulating progesterone concentrations were low. In cows and goats that had secretion regularly removed from the mammary glands late in gestation, the onset of triglyceride extraction occurred up to several days before parturition, when circulating progesterone concentrations were moderately high (up to 3.9 ng/ml in the cow and 6.2 ng/ml in the goat).