脑组织特异性表达胰岛素样生长因子-1转基因小鼠制备

目的制备脑组织特异性表达胰岛素样生长因子-1(Insulin-Like Growth Factors 1,IGF-1)转基因小鼠。方法采用精子为载体法进行基因转导,出生后小鼠PCR检测,建立转基因小鼠系,用Western blot和免疫荧光法分别检测转基因小鼠海马齿状回亚粒状区(subgranular zone,SGZ)IGF-1表达量和报告基因EGFP阳性细胞数。结果获得3只阳性小鼠,2只外源基因能稳定遗传,并建立了转基因小鼠系,转基因鼠SGZ区域IGF-1表达量显著高于正常鼠,EGFP也在此区域表达。结论成功制备IGF-1转基因小鼠。     

Tet-on系统诱导乳腺特异表达CUEDC2转基因小鼠的制备

目的：建立Tet-on系统诱导乳腺特异表达CUEDC2的转基因小鼠模型。方法：构建转基因表达载体,经细胞诱导表达验证后,酶切得到含有CUEDC2的线性DNA片段,并采用显微注射的方法及后续筛选鉴定,得到CUEDC2转基因小鼠。进而通过与乳腺特异表达的MMTV-rtTA转基因小鼠交配,得到CUEDC2/rtTA双阳性转基因小鼠。利用2mg/ml的多西环素（Doxycycline）诱导小鼠体内CUEDC2表达,通过Western Blot、免疫组化检测表达。结果：经诱导,CUEDC2/rtTA双阳性转基因小鼠的2个Founder的F1、F2代在转录水平和蛋白水平均成功表达CUEDC2, 并具有乳腺特异性。结论：Tet-on系统诱导乳腺特异表达CUEDC2的转基因小鼠制备成功.     

用精原干细胞介导法制备乙肝病毒X基因转基因小鼠的实验研究

目的　探讨精原干细胞介导法制备乙肝病毒X基因转基因小鼠的可行性 .方法　构建乙肝病毒X基因表达载体 pcDNA3 X ;应用微量注射针将脂质体包裹的 pcDNA3 X表达载体直接注入C5 7BL/ 6N雄性小鼠睾丸的曲细精管内 ,于第一次注射后 6周 ,与雌鼠交配 ,用PCR法和免疫组化法检测仔鼠基因组中的外源DNA和肝组织中表达的 pX蛋白 .结果　共产仔鼠 16只 ,其中 1只为阳性仔鼠 ,阳性率为 6 .2 5 %.结论　初步明用精原干细胞介导法制备X基因转基因小鼠是可行的 .     

全身性过表达和表皮特异性过表达胸腺素β4小鼠皮肤中目的基因表达差异分析

胸腺素β4(Thymosinβ4,Tβ4)能够促进毛发生长.旨在构建CAG启动子指导的全身性过表达Tβ4小鼠(CTP小鼠)和角蛋白14(Keratin14,Krt14)启动子指导的表皮特异性过表达Tβ4小鼠(KTP小鼠),比较两种转基因小鼠模型的Tβ4表达情况,为进一步研究Tβ4对毛发生长的影响以及其作用机制奠定基础.首先构建CAG启动子指导的全身性过表达载体pODsRed-CTP,然后用pODsRed-CTP和Krt14启动子指导的pODsRed-KTP表达载体,通过原核注射技术制作转Tβ4基因小鼠.PCR鉴定得到CTP小鼠2只,阳性率1.6%;KTP小鼠4只,阳性率8%.通过Real-time PCR检测Tβ4基因mRNA水平表达量,结果显示Tβ4在CTP小鼠皮肤中mRNA水平表达量是对照小鼠的1.24和1.13倍,在KTP小鼠皮肤中mRNA水平表达量分别是对照小鼠的4.22、1.84、4.60和2.74倍.Western blotting检测Tβ4在蛋白水平的表达,发现CTP小鼠皮肤中Tβ4蛋白的表达量没有显著变化,而KTP小鼠皮肤中Tβ4蛋白表达水平显著升高.与CTP小鼠相比,KTP小鼠更适合作为Tβ4过表达小鼠模型,深入研究Tβ4对毛发的影响以及作用机制.     

转基因小鼠乳腺表达人乳过氧化物酶的初步研究

从人基因组PAC文库中筛选出人乳过氧化物酶（hLPO）基因,利用长片段PCR的方法获得hLPO基因5’端约3kb片段,通过酶切方法获得hLPO基因3’端约27kb片段,将这两部分拼接并克隆到乳腺特异性表达载体pBC1上,构建以山羊β-casein启动区指导的hLPO的转基因表达载体pBC1-hLPO。利用显微注射的方法获得28只FO代小鼠,经PCR检测和Southerm杂交分析证实,有5只小鼠（4♂,1♀）为整合hLPO基因的转基因阳性小鼠,整合率为17.86％,整合拷贝数在1至5之间。利用SDS-PAGE凝胶电泳和Western blot印迹分析FO、F1代共3只雌性转基因小鼠乳样,结果表明hLPO重组蛋白的特异条带不明显。     

Plcd1转基因小鼠的制备

通过制备Plcd1转基因小鼠研究Plcd1基因功能.首先逆转录PCR获得约2.2kb的Plcd1基因全长,T载体克隆后测序验证;以pMD19-T-Plcd1为模板,通过设计引物及PCR扩增引入酶切位点,与pEF6/V5-His同时进行酶切、连接,构建pEF6/V5-His-Plcd1表达载体;经真核表达验证后,酶切获得目标片段,显微注射681枚受精卵,在82只仔鼠中获得转基因阳性首建鼠15只,其中13只稳定遗传并建系.PLCD1-HIS融合蛋白在睾丸组织中表达,在皮肤组织中无表达.Plcd1转基因小鼠为Plcd1功能研究奠定了基础.     

人溶菌酶基因密码子优化及其在小鼠乳腺中的表达

人溶菌酶是人体内的一种非特异性免疫物质,具有杀菌消炎、免疫调节、改善胃肠道菌群等作用,在抗生素替代方面具有广阔的应用前景。已报道人溶菌酶基因c DNA在内源的αS2酪蛋白基因启动子调控下的表达量不高(23~31μg/m L),特尝试密码子优化策略以改善表达量。优化了人溶菌酶基因c DNA的密码子,连入以小鼠乳清酸蛋白(m WAP)基因座序列为调控元件的乳腺表达载体,验证其表达效率。通过原核注射制备转基因小鼠,PCR检测发现,出生的21只小鼠中有4只为转基因阳性,且能按孟德尔遗传规律传递给下一代;Western blot检测表明,小鼠乳汁中有微量的重组人溶菌酶表达。因此,密码子优化后的人溶菌酶基因可以在小鼠乳腺中获得有效表达,这可为外源基因定点整合的人溶菌酶转基因动物的高效表达研究奠定基础。     

MicroRNA-122过表达转基因小鼠的建立

目的:建立microRNA-122 (mir-122)过表达转基因小鼠模型,为今后研究肝脏发育,分化及肝癌预防、诊断及治疗等提供实验模型.方法:构建mir-122过表达载体,利用受精卵前核显微注射法将mir-122过表达载体导入小鼠受精卵中,获得mir-122过表达转基因小鼠.PCR法检测转基因小鼠的整合情况,实时定量PCR法检测mir-122过表达转基因小鼠肝脏及其主要器官mir-122及其确定靶基因高亲和性阳离子氨基酸转运体-1(CAT-1),切割同源物1(CUTL-1),血清反应因子(SRF)的表达情况.结果:mir-122过表达转基因小鼠,与同窝阴性鼠相比,mir-122在过表达转基因小鼠肝脏及主要器官中的表达升高,mir-122靶基因表达水平下降.结论:成功构建mir-122过表达转基因小鼠.     

基于Cre-LoxP系统的条件性定点敲入人源hRas基因小鼠的建立

目的 构建基于Cre- LoxP系统的条件性定点敲入人源hRas基因(c-Ha-ras)的小鼠,以获得hRas基因在特定组织器官条件性表达的小鼠模型,用于药物的临床前致癌性安全评价及相关机制研究。方法 首先构建hRas打靶载体,电击法转染入小鼠胚胎干细胞(ES细胞),用正负法筛选阳性ES细胞,通过PCR、 Southern鉴定后,将正确重组hRas基因的ES细胞导入C57BL/6 J小鼠囊胚,移入同步发育的受体鼠子宫,妊娠足月出生的嵌合体小鼠与C57BL/6 J小鼠交配获得杂合子hRasfl/+小鼠。再将杂合子hRasfl/+小鼠间交配获得纯合子小鼠hRasfl/fi,然后与全身组织细胞表达Cre重组酶的Tg (EIIa-cre)小鼠进行交配,获得全身细胞表达hRas基因的hRas-EIIa-cre小鼠,并通过荧光定量PCR方法检测不同日龄胚胎期仔鼠的hRas基因表达水平。结果 成功构建了基于Cre- LoxP系统的人源hRas基因条件性定点敲入小鼠模型的载体,筛选得到的一个正确克隆,电转ES细胞后进行Southern blot鉴定,经过初筛和复筛,共获得12个阳性克隆,挑选A11号克隆ES细胞进行囊胚注射,移植了48枚胚胎,出生9只小鼠,其中6只为嵌合鼠,将嵌合率>50%的雄鼠与野生型C57BL /6 J雌鼠进行交配,出生21只后代,其中鉴定有4只hRasfl/+小鼠;hRasfl/+小鼠间交配出生29只小鼠,其中有14只纯合子hRasfl/fi小鼠;hRasfl/fl小鼠与Tg (EIIa-cre)工具鼠交配6次,未有仔鼠出生;跟踪不同发育时期胚胎中hRas基因的表达发现,E10.5～15.5 d胚胎中检测到hRas基因表达。结论 成功建立运用Cre- LoxP系统建立了带有hRas基因敲入的纯合子小鼠hRasfl/fl,未能得到全身细胞高效表达人源hRas基因的hRas-EIIa-cre小鼠,但为进一步利用hRasfl/fl小鼠模型建立其他组织特异性的条件性基因敲入小鼠模型做好技术储备。     

基于Cre／Loxp系统Tyr03、Axl受体酪氨酸激酶转基因小鼠的建立

采用大肠杆菌β一半乳糖酶基因LacZ作为报告基因,建立Tyr03、Axl受体酪氨酸激酶含有loxp位点的转基因小鼠。与不同组织特异性Cre小鼠杂交,获得时空性表达Tyr03、Axl的转基因小鼠,为研究其在各个组织中的功能奠定基础。方法构建含有loxp位点的Tyr03、Axl受体酪氨酸激酶质粒,测序正确后,通过显微注射法将插入CAG启动子下游的Geo—STOP—msAxl及Geo—STOP—msTyr03基因的转基因载体注射入BDFI供体鼠受精卵,移植受精卵至ICR受体鼠,待产仔后,通过双引物PCR鉴定F。以及Fn代小鼠的基因型,RT—PCR,X—gal染色观察小鼠组织中LaeZ基因,运用WB（WesternBlot）检测转基因小鼠和野生型小鼠体内目的蛋白表达的差异。结果获得了含有目的基因的转基因阳性小鼠;通过RT—PCR对阳性小鼠证明LacZ基因的存在;对存在LacZ基因的小鼠进行X—gal染色,在Geo—STOP—msTyr03的脑、睾丸、胸腺中观察到蓝色沉淀,在Geo—STOP—msAxl的脑、心、肾中观察到蓝色沉淀,间接说明目的基因能表达的组织和器官;对双阳性的Geo．STOP．msTyr03、Geo．STOP—msAxl转基因小鼠和野生型小鼠,WB检验证实Tyr03、Axl基因的表达没有差异,说明在加入stop序列之后,Tyr03基因确实存在,而且表达受到抑制。结论成功建立Geo-STOP—msAxl及Geo—STOP—msTyr03酪氨酸受体转基因小鼠。     

Expression of recombinant human lysozyme in the milk of transgenic mice

Human lysozyme is a 130-aa (amino acid) alkaline polypeptide, and has both anti-bacterial and anti-viral properties which make it an important component of human natural immunity system. As a first step toward the ultimate goal of improving the anti-bacterial properties of bovine and ovine milk, a transgenic mouse that contains the genomic DNA sequence of the human lysozme gene has been generated for the first time. From 83 mice generated by microinjection, a total of 6 positive transgenic mice were identified by PCR and Southern blot. F1 mice positive for transgene in lines were also detected by PCR. This shows that transgene could be transmitted from founder transgenic mice to their offspring. Recombinant human lysozyme (rHlys) was found in the whey of 3 female positive transgenic mice by Western blot. The highest concentration of rHlys for transgenic mice was 0.2mg/mL. The antibacterial activity of the whey for transgenic mice was highly enhanced up to 0.4 times as much as that of human, while that of non-transgenic mouse was very low. Although the lysozyme activity of transgenic mice is still lower than that of human, the rHlys exhibits the same specific activity as that of human lysozyme. It provides a strong basis for further studies into the possible application of rHlys express in mammary gland.     

High expression of human serum albumin in milk of trans- genie mice directed by the goat p-casein gene promoter region

We have constructed a mammary gland expression vector that contained the goat β-casein gene pro-moter, 5'upstream regulatory region, exons 1, 2, intron 1 as well as the human serum albumin (hALB) mini-gene (including the full-long sequences of hALB cDNA and its intron 1). Injection of the vector into mouse tail veins showed that the recombinant construct was expressed only in mammary glands. The vector was microinjected into the mouse fertilized eggs, followed by transferring the eggs into the foster mice. 33 F0 mice were obtained. Of the 33, 8 mice (5 , 3 ) were transgenic with hALB gene integration identified by PCR as well as Southern blot hybridization. The integration rate was 24.2% (8/33). Western blot analysis showed that 3 female transgenic mice had hALB expression in their milk. The hALB contents in milk reached 3.54, 0.21 and 3.03 g/L, respectively.     

iRhom2Uncv 小鼠乳腺发育障碍的转录组分析

摘要:目的 研究 iRhom2Uncv 小鼠乳腺表型相关的差异表达基因,为深入研究 iRhom2Uncv 小鼠乳腺发育障碍的相关机制提供靶点信息。 方法 对 12 周龄的野生型和 iRhom2Uncv 小鼠的乳腺组织进行 whole-mounts 染色,分析乳腺发育形态,收集乳腺组织进行转录组测序,筛选差异表达基因,ELISA 检测血清激素水平。 结果 与野生型小鼠相比,iRhom2Uncv 小鼠乳腺侧枝导管较稀疏,差异最显著的 18 个基因中有 6 个在 iRhom2Uncv 小鼠中表达上调,12 个表达下调。 其中 3 个催乳素家族基因在 iRhom2Uncv 小鼠中不表达,且在血清中几乎检测不到催乳素表达,而血清中的雌激素水平相比野生型小鼠较高。 结论 iRhom2Uncv 突变的小鼠存在乳腺发育障碍,可能与 iRhom2Uncv 小鼠中催乳素和 Zfp281 的缺失表达有关,生物信息学分析发现,iRhom2Uncv 小鼠神经发育可能存在不同于野生型小鼠的表型。     

Building of hFⅨ transgenic mice by spermatogenic cells

Human FⅨ expression vector pCMVⅨ was packaged by effectene^TM reagent and injected into mice seminiferous tubules with glass pipettes.The expressional frame of pCMVⅨ was examined by PCR and Southern blotting among 41 progenies.There were 2(4%) mice being integrated with hFⅨ gene into chromosomes.4.6ng/mL of hFⅨ protein was expressed in plasma of one mouse,which was tested by ELISA.We demonstrated that building of transgenic animals by spermatogonial stem cells is an efficient method.Meanwhile,it has also been proved to be an alternative choice for mammary gland bioreactor.     

High expression of human serum albumin in milk of transgenic mice directed by the goat β-casein gene promoter region

We have constructed a mammary gland expression vector that contained the goat β-casein gene promoter, 5′upstream regulatory region, exons 1,2, intron 1 as well as the human serum albumin (hALB) mini-gene (including the full-long sequences of hALB cDNA and its intron 1). Injection of the vector into mouse tail veins showed that the recombinant construct was expressed only in mammary glands. The vector was microinjected into the mouse fertilized eggs, followed by transferring the eggs into the foster mice. 33 F₀ mice were obtained. Of the 33, 8 mice (5♀, 3 ♂) were transgenic with hALB gene integration identified by PCR as well as Southern blot hybridization. The integration rate was 24.2% (8/33). Western blot analysis showed that 3 female transgenic mice had hALB expression in their milk. The hALB contents in milk reached 3.54, 0.21 and 3.03 g/L, respectively.     

外源基因在转基因动物乳腺中的特异性表达研究

动物基因工程研究最大的突破就是转基因动物研究的进展,自八十年代初第一批转基因小鼠问世以来,转基因动物的研究已从方法学研究步入了应用性研究阶段,转基因动物除作为研究工具广泛应用于发育生物学、免疫学、遗传学以及医学等生命科学领域外,外源基因在转基因动物的特异性表达,尤其是在乳腺的表达,又可将转基因用作生物反应器进行了生物活性蛋白的生产而于商业生产,在转基因动物乳腺的特异性表达研究中,寻找乳蛋白基因调控     

Correction of RNA aberrant splice increases foreign gene expression in transgenic mice

Transgenic mice with mammary gland secreting human granulocyte colony stimulating factor (G-CSF) were produced using mice whey acid protein gene promoter. It was found that there was very low expression level in mammary gland. Human G-CSF cDNA was obtained by RT-PCR from transgenic mice mammary gland. Sequence analysis showed that this G-CSF gene deleted the 4th exon, and compared with human G-CSF genomic DNA, there were donor and acceptor splice sites in the deletion fragment. It was considered that the 3rd and 4th introns also delete in G-CSF fragment. The transgenic construct was corrected by deleting the 3rd and 4th introns to construct the minigene, which was used to produce transgenic mice by microinjection. Northern blot showed that G-CSF expression using the new construct increased 5.4 times as that before in transgenic mice. The results suggested that it was possible that RNA aberrant splice result in low expression in transgenic mice.     

Alteration of the quality of milk by expression of sheep beta-lactoglobulin in transgenic mice

Milk contains a large amount of protein, most of which consists of a few major species synthesized in the mammary gland. The genes encoding these proteins are single-copy, and expressed during pregnancy and lactation. Although beta-lactoglobulin (BLG) is the major protein in the whey of ruminants, it is not present in rodent milk. We have generated transgenic mice carrying the sheep BLG gene, and show that in such mice, BLG is specifically and abundantly expressed in the mammary gland during lactation. This results in a remarkable alteration of milk composition. These findings suggest that the manipulation of milk composition by gene transfer has considerable potential for the improvement of dairy animals.     

RANK ligand mediates progestin-induced mammary epithelial proliferation and carcinogenesis

RANK ligand (RANKL), a TNF-related molecule, is essential for osteoclast formation, function and survival through interaction with its receptor RANK. Mammary glands of RANK- and RANKL-deficient mice develop normally during sexual maturation, but fail to form lobuloalveolar structures during pregnancy because of defective proliferation and increased apoptosis of mammary epithelium. It has been shown that RANKL is responsible for the major proliferative response of mouse mammary epithelium to progesterone during mammary lactational morphogenesis, and in mouse models, manipulated to induce activation of the RANK/RANKL pathway in the absence of strict hormonal control, inappropriate mammary proliferation is observed. However, there is no evidence so far of a functional contribution of RANKL to tumorigenesis. Here we show that RANK and RANKL are expressed within normal, pre-malignant and neoplastic mammary epithelium, and using complementary gain-of-function (mouse mammary tumour virus (MMTV)-RANK transgenic mice) and loss-of function (pharmacological inhibition of RANKL) approaches, define a direct contribution of this pathway in mammary tumorigenesis. Accelerated pre-neoplasias and increased mammary tumour formation were observed in MMTV-RANK transgenic mice after multiparity or treatment with carcinogen and hormone (progesterone). Reciprocally, selective pharmacological inhibition of RANKL attenuated mammary tumour development not only in hormone- and carcinogen-treated MMTV-RANK and wild-type mice, but also in the MMTV-neu transgenic spontaneous tumour model. The reduction in tumorigenesis upon RANKL inhibition was preceded by a reduction in pre-neoplasias as well as rapid and sustained reductions in hormone- and carcinogen-induced mammary epithelial proliferation and cyclin D1 levels. Collectively, our results indicate that RANKL inhibition is acting directly on hormone-induced mammary epithelium at early stages in tumorigenesis, and the permissive contribution of progesterone to increased mammary cancer incidence is due to RANKL-dependent proliferative changes in the mammary epithelium. The current study highlights a potential role for RANKL inhibition in the management of proliferative breast disease.