Tetracycline-dependent expression of the human erythropoietin gene in transgenic chickens

A critical problem in the production of transgenic animals is the uncontrolled constitutive expression of the foreign gene, which occasionally results in serious physiological disorders in the transgenic animal. In this study, we report successful production of transgenic chickens that express the human erythropoietin (hEPO) gene under the control of a tetracycline-inducible promoter. A recombinant Moloney murine leukemia virus (MoMLV)-based retrovirus vector encapsidated with vesicular stomatitis virus G glycoprotein (VSV-G) was injected beneath the blastoderm of unincubated chicken embryos (stage X). Out of 198 injected eggs, 15 chicks hatched after 21 days of incubation and 14 hatched chicks expressed the vector-encoded hEPO gene when fed doxycycline, a tetracycline derivative, without any significant physiological dysfunctions. The expression of hEPO reverted to the pre-induction state by removing doxycycline from the diet. The biological activity of the hEPO produced in the transgenic chickens was comparable to commercially available CHO cell-derived hEPO. Successful germline transmission of the transgene was also confirmed in G1 transgenic chicks produced from crossing G0 transgenic roosters with non-transgenic hens. Tetracycline-inducible expression of the hEPO gene was also confirmed in the blood and eggs of the transgenic chickens.     

Tetracycline-inducible gene expression in nuclear transfer embryos derived from porcine fetal fibroblasts transformed with retrovirus vectors

A critical problem of transgenic livestock production is uncontrollable constitutive expression of the foreign gene, which usually results in serious physiological disturbances in transgenic animals. One of the best solutions for this problem may be use of controllable gene expression system. In this study, using retrovirus vectors designed to express the enhanced green fluorescent protein (EGFP) gene under the control of the tetracycline-inducible promoter, we examined whether the expression of the transgene could be controllable in fibroblast cells and nuclear transfer (NT) embryos of porcine. Transformed fibroblast cells were cultured in medium supplemented with or without doxycycline (a tetracycline analog) for 48 hr, and the induction efficiency was measured by comparing EGFP gene expression using epifluorescence microscopy and Western and Northern blot analyses. After the addition of doxycycline, EGFP expression increased up to 17-fold. The nuclei of transformed fibroblast cells were transferred into enucleated oocytes. Fluorescence emission data revealed strong EGFP gene expression in embryos cultured with doxycycline, but little or no expression in the absence of the antibiotic. Our results demonstrate the successful regulation of transgene expression in porcine nuclear transfer embryos, and support the application of an inducible expression system in transgenic pig production to solve the inherent problems of side-effects due to constitutive expression of the transgene.     

一种高效的单质粒模式四环素诱导表达系统的构建

现有的四环素诱导调控系统基于两个单独的质粒分别表达反式结合蛋白和外源基因.其缺点是在建立转基因定量表达动物模型时,需要制备和维持两个动物品系,再进行杂交才有可能获得双转基因后代,步骤繁琐,难度较大.针对上述缺陷,本研究尝试将反式蛋白rtTA表达框和低背景响应元件Ptight组装到同一个载体上,构建为严谨型单载体模式的诱导表达系统pTRE-Tight-rtTA,并通过两种报告基因的表达对其调控活性进行了研究.含有荧光素酶和绿色荧光蛋白的pTRE-Tight-rtTA-Luc和pTRE-Tight-rtTA-EGFP报告载体分别转染猪肾PK15细胞并经强力霉素处理,均可成功诱导报告基因的定量表达.在等摩尔转染条件下,单载体系统的诱导效率明显高于双载体系统（Dox-1 000 ng,10 倍;Dox-10 000 ng,8 倍）.该诱导型单载体系统的成功构建为外源基因的定量表达提供了新手段,为转基因定量表达动物模型的研究提供了新策略.     

Production of transgenic chickens constitutively expressing human erythropoietin (hEPO): Problems with uncontrollable overexpression of <Emphasis Type="Italic">hEPO</Emphasis> gene

The chicken is a promising candidate as a bioreactor for the economical mass production of human therapeutic proteins. Here, we report the successful generation of transgenic chickens that produce high concentrations of human erythropoietin (hEPO) in the blood. Using a Moloney murine leukemia virus (MoMLV)-based pseudotyped retrovirus vector packaged with vesicular stomatitis virus G glycoprotein (VSV-G), the hEPO gene under the control of cytomegalovirus (CMV) promoter was introduced to the blastoderm of freshly laid chicken eggs (stage X). Out of 200 injected eggs, 12 chicks were hatched after 21 days of incubation, and all of the G₀ hatched chicks expressed the vector-encoded hEPO gene. One of the G₀ roosters successfully transmitted the hEPO gene to its G₁ progeny by crossing with non-transgenic hens. The concentration of hEPO protein in the chicken blood serum was as high as 90 μg/mL. Although humans and chickens belong to different classes of the phylogenetic tree, human EPO caused devastating problems in transgenic chickens, including sudden death, polycythemia, vasodilation, and so on, which may be due to the uncontrolled constitutive expression of exogenous protein in the chicken body. Despite many disorders, however, we were able to generate chicks of G₂ generation sired by a rooster of G₁ generation confirming successful establishment of a new line of transgenic chicken characterized by high expression of the hEPO gene. With these chickens, we believe that studies on the evaluating the possibilities of the transgenic animal-mediated bio-pharming and on the hEPO-induced physiological side effects will be greatly facilitated.     

Construction of an inducible expression shuttle vector for Laribacter hongkongensis, a novel bacterium associated with gastroenteritis

An Escherichia coli-Laribacter hongkongensis shuttle vector (pPW380) was constructed by ligating the 4701-bp EcoRI digested fragment of pHLHK8 to EcoRI digested pBK-CMV. An E. coli-L. hongkongensis inducible expression shuttle vector was further constructed by ligating a 2105-bp fragment that contains the tetracycline repressor and tetracycline-inducible promoter region of pALC2084 to the 8897-bp fragment of pPW380, deletion of the green fluorescent protein gene, and insertion of a multiple cloning site. This inducible expression system was able to express two commonly used reporter genes, the green fluorescent protein gene and the glutathione S-transferase gene, efficiently in E. coli and L. hongkongensis.     

Dual promoter expression system with insulator ensures a stringent tissue-specific regulation of two reporter genes in the transgenic fish

The precise control of spatiotemporal expression of target genes is crucial when establishing transgenic animals, and the introduction of genes for fluorescent marker proteins is inevitable for accelerating research at molecular levels. To assist this, we constructed a novel dual promoter expression vector for two independent reporter genes, green fluorescent protein (GFP) and red fluorescent protein (mCherry). Their expression is designed under the control of two distinct tissue-specific promoters, e.g. zebrafish cardiac muscle-specific promoter (cmlc2) and medaka skeletal muscle-specific promoter (myl2) derived from the myosin light chain 2 genes, and they are placed in a head-to-head orientation. After microinjecting the dual promoter expression vector into fertilized eggs of medaka, the developing fish embryos and the resulting transgenic fish lines showed strong GFP signal in the whole body (skeletal muscle) and mCherry signal in the heart (cardiac muscle). However, weak GFP signal was observed in the heart, indicating a leakiness of the skeletal muscle promoter. To improve the stringency of dual promoter expression, we inserted two chicken-derived insulators, e.g. tandem copies of the core sequence (250 bp) of cHS4 (5′-hypersensitive site-4 chicken beta-globin insulator), in the boundary of two promoters. The dual promoter expression vector with insulator now ensured the stringent tissue-specific expression in the transgenic fish lines. Thus, our dual promoter expression system with insulator is compatible to the conventional IRES and fused reporter gene systems and will be an alternative method to produce the transgenic fishes.     

CpG methylation modulates tissue-specific expression of a transgene in chickens

The use of genetically modified germ cells is an ideal system to induce transgenesis in birds; the primordial germ cell (PGC) is the most promising candidate for this system. In the present study, we confirmed the practical application of this system using lentivirus-transduced chicken gonadal PGCs (gPGCs). Embryonic gonads were collected from 5.5-d old Korean Oge chickens (black feathers). The gPGC population was enriched (magnetic-activated cell sorting technique) and then they were transduced with a lentiviral vector expressing enhanced green fluorescent protein (eGFP), under the control of the Rous sarcoma virus (RSV) promoter. Subsequently, the eGFP-transduced PGCs were transplanted into blood vessels of 2.5-d-old embryonic White Leghorn (white feathers). Among 21 germline chimeric chickens, one male produced transgenic offspring (G₁ generation), as demonstrated by testcross and genetic analysis. A homozygous line was produced and maintained through the G₃ generation. Based on serum biochemistry, there were no significant physiological differences between G₃ homozygotes and non-transgenic chickens. However, since eGFP transgene expression in G₃ chickens varied among tissues, it was further characterized by Western blotting and ELISA. Furthermore, there were indications that DNA methylation may have affected tissue-specific expression of transgenes in chickens. In conclusion, the PGC-mediated approach used may be an efficient tool for avian transgenesis, and transgenic chickens could provide a useful model for investigating regulation of gene expression.     

Germ line transformation of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>, using the transposable element <Emphasis Type="Italic">Minos</Emphasis>

We investigated the use of Minos as a vector for transgenesis in the silkworm, Bombyx mori. We first constructed a vector plasmid with the green fluorescent protein (GFP) gene fused with the silkworm cytoplasmic actin gene (A3) promoter, and a helper plasmid with the Minos transposase gene controlled by the same A3 promoter. Injection of the vector and helper plasmid DNA into silkworm eggs produced transgenic animals in the following generation. The efficiency of transgenic silkworm production using this method was much lower than that obtained using piggyBac-mediated germ line transformation. However, >40-fold increase in the efficiency of producing transgenic silkworms was obtained using an in vitro synthesized source of Minos transposase mRNA. We conclude that the Minos transposon is a useful vector for construction of transgenic silkworms, particularly when in vitro synthesized mRNA is used. This is the first report showing that Minos can be used as a vector for germ-line transformation in lepidopteran insects.     

Retrovirus-mediated gene transfer and expression of EGFP in chicken

Here, we successfully demonstrate expression of the EGFP (enhanced green fluorescence protein) gene in chickens using replication-defective MLV (murine leukemia virus)-based retrovirus vectors encapsidated with VSV-G (vesicular stomatitis virus G glycoprotein). The recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). After 12 days incubation, all of the eight living embryos assayed were found to express this vector-encoded EGFP gene, which was under the control of the RSV (Rous Sarcoma Virus) promoter, in diverse organ tissues, including head, beak, neck, wing, hock, tail, toes, heart, amnion, and yolk sac. Surprisingly, despite the presumed cytotoxicity of EGFP, some embryos hatched and survived and these had prominent green fluorescent spots, both in internal organs and externally.     

荧光转基因斑马鱼Tg(ttn.2:EGFP)的构建

为了建立一种用于研究肌肉和心脏发育及其相关疾病的绿色荧光蛋白(enhanced green fluorescent protein,EGFP)转基因斑马鱼品系,本研究使用斑马鱼ttn.2基因编码区上游启动子序列和绿色荧光蛋白基因编码序列构建了重组表达载体,并将该载体和Tol2转座酶的加帽mRNA显微共注射入斑马鱼1-细胞期胚胎,通过荧光检测、遗传杂交筛选和分子鉴定等方法,成功建立了能稳定遗传的Tg(ttn.2:EGFP)转基因斑马鱼品系。荧光表达分析及原位杂交分析结果表明,绿色荧光信号在斑马鱼肌肉和心脏组织中特异表达模式与ttn.2基因的mRNA表达一致。通过反向PCR鉴定转基因表达载体在F1代斑马鱼品系中的随机整合位点,结果表明:No.33转基因品系的EGFP基因整合在斑马鱼的4号和11号染色体上,No.34转基因品系则整合在1号染色体上。该荧光转基因斑马鱼品系Tg(ttn.2:EGFP)的成功构建为肌肉和心脏发育以及相关疾病研究提供了一个新的理想实验模型。此外,绿色荧光强烈表达的斑马鱼品系还可以作为一种新的观赏鱼。     

Expression of green fluorescent protein in oligodendrocytes in a time- and level-controllable fashion with a tetracycline-regulated system

Developments in transgenic technology have greatly enhanced our ability to understand the functions of various genes in animal models and relevant human diseases. The tetracycline (tet)-regulated transactivation system for inducing gene expression allowed us to control the expression of exogenous genes in a temporal and quantitative way. The ability to manipulate a cell-specific promoter enabled us to express one particular protein in a single type of cell. The combination of a tetracycline system and a tissue-specific promoter has led us to the development of an innovative gene expression system, which is able to express genes in a cell type-specific and time- and level-controllable fashion. An oligodendrocyte-specific myelin basic protein (MBP) gene promoter controls the reversed tet-inducible transactivator. The green fluorescent protein (GFP) gene was placed under the control of the human cytomegalovirus (CMV) basic promoter in tandem with seven tet-responsive elements (TRE), binding sites for the activated transactivator. Upon the addition of doxycycline (DOX, a tetracycline derivative), tet transactivators became activated and bound to one or more TRE, leading to the activation of the CMV promoter and the expression of GFP in oligodendrocytes. We have successfully expressed GFP and luciferase at high levels in oligodendrocytes in a time- and dose-dependent fashion. In the absence of DOX, there was almost no GFP expression in oligodendroglial cultures. Graded levels of GFP expression were observed after induction with DOX (0.5 to 12.5 microg/ml). Our data indicate that this inducible gene expression system is useful for the study of gene function in vivo and for the development of transgenic animal models relevant to human diseases such as multiple sclerosis.     

心脏特异表达绿色荧光斑马鱼模型的建立与评估

本课题组前期研究中, 利用斑马鱼cmlc2 (Cardiac myosin light chain 2)基因启动子构建了一个用于斑马鱼心脏组织特异表达外源基因的转基因表达载体pTol2-cmlc2-IRES-EGFP。文章利用该载体构建了一个稳定表达EGFP的转基因斑马鱼品系, 并初步分析了EGFP的表达对该转基因斑马鱼品系的心脏发育和功能的影响。结果表明, 在建立的转基因斑马鱼品系早期胚胎发育过程中, 绿色荧光信号在心脏中特异表达, 该表达模式与原位杂交分析的cmlc2的表达模式结果相同; 该转基因斑马鱼品系的心脏形态及发育生长正常; 进一步通过M-Mode分析心脏生理学功能的结果表明：该转基因品系心动周期、心率、收缩与舒张表面积及表面积缩短率等重要生理指标与正常野生型的斑马鱼对照组相比没有显著差别。以上结果表明该转基因品系中绿色荧光蛋白的表达对斑马鱼心脏的发育和功能没有影响。研究结果为进一步利用该载体建立外源目的基因转基因表达模型, 研究心脏表达基因的功能奠定了重要基础。     

转基因鸡生物反应器载体的构建及其表达特性分析

以增强型绿色荧光蛋白和萤火虫荧光素酶为报告基因,构建了鸡卵清蛋白启动子表达载体和慢病毒载体,以巨细胞病毒 (Cytomegalovirus,CMV)启动子表达载体为对照,转染或感染鸡原代输卵管上皮细胞、鸡胚成纤维细胞、鼠3T3-L1前脂肪细胞和牛乳腺上皮细胞,通过荧光和酶活性检测,旨在筛选出用于实现转基因鸡生物反应器的高效特异性表达载体。结果发现,鸡卵清蛋白启动子表达载体转染以上4种细胞后2种标记基因均有表达,没有表现出明显的细胞特异性,且荧光素酶检测结果表明其在各细胞组中表达活性都低于CMV启动子表达载体100倍以上;慢病毒载体感染以上4种细胞后2种标记基因均有表达,在鸡输卵管上皮细胞组感染单个细胞的病毒颗粒 (Multiplicity of infection,MOI) 为20时绿色荧光蛋白表达量就可以达到CMV启动子表达载体的水平。上述结果表明,基于卵清蛋白基因调控序列构建的表达载体无法实现外源基因的高效、特异性表达,而慢病毒载体在表达活性和广泛性上可以用于进行鸡输卵管生物反应器的研究。     

Tetracycline-inducible gene expression in mycobacteria within an animal host using modified Streptomyces tcp830 regulatory elements

Inducible expression systems are powerful tools for studying gene function. Though several inducible expression systems are now available for mycobacteria, none have been used to modulate bacterial gene expression during an animal infection. A tetracycline-inducible expression system from Streptomyces coelicolor was successfully adapted for use in mycobacteria. To prevent baseline expression without induction, S. coelicolor tetR gene was overexpressed using the acetamidase promoter and regulatory gene block. Target gene expression was controlled by the S. coelicolor tcp830 promoter and operator allele. The −10 promoter consensus sequence of the tcp830 promoter was modified to better resemble known strong mycobacterial promoters. Using this system, induction of tetR fully repressed tcp830-dependent expression of green fluorescent protein (GFP) to baseline levels. Addition of anhydrotetracycline led to a 62-fold induction of GFP expression in vitro and 15-fold induction in a mouse mycobacterial peritonitis model in the presence of maximal tetR expression. Chemically regulatable gene expression during animal infection may be a useful tool in studying mycobacterial pathogenesis.     

绿色荧光蛋白基因原核表达载体的构建及其在昆虫肠道短短芽孢杆菌和苏云金芽孢杆菌中的表达

【目的】构建带有苏云金芽孢杆菌cry3a基因非芽孢依赖启动子和绿色荧光蛋白基因gfp(Green Fluorescent Protein)的原核表达载体,并转化从桑粒肩天牛幼虫肠道分离的两株常驻细菌短短芽孢杆菌CQUBb和苏云金芽孢杆菌CQUBt,以检测cry3a启动子在昆虫肠道常驻菌中的启动子活性,获得GFP标记菌株,为常驻菌在昆虫幼虫肠道中的定殖情况和杀虫工程菌的构建奠定基础。【方法】采用重叠延伸PCR将cry3a基因启动子和gfp基因进行融合,并与pHT304载体连接构建重组质粒pHT3AG,获得的重组质粒以电脉冲转化肠道常驻菌短短芽孢杆菌CQUBb和苏云金芽孢杆菌CQUBt,于可见光和荧光显微镜下观察荧光并通过SDS-PAGE分析重组菌株的蛋白表达情况,然后对重组菌株进行生长动力学分析和稳定性测试。【结果】重组菌在营养期大量组成型表达GFP,经电泳分离在凝胶上出现约29kDa的特异蛋白条带;重组菌生长曲线与出发菌没有显著差异,说明外源质粒未对宿主菌的生长带来明显不利影响;抗性条件下传代30次后两菌株外源质粒稳定性仍可达95%、67%;两个菌株比较,CQUBb比CQUBt质粒转化率高、重组菌GFP表达时间长、表达量大,并且重组菌株稳定性好。【结论】成功地将cry3a基因核心启动子和gfp基因转入桑粒肩天牛幼虫肠道常驻菌,实现了该启动子在Bt之外的菌株中发挥作用,构建了两个GFP标记菌株;重组基因工程菌株表达量大,稳定性好,可以用作昆虫肠道内微生态研究和芽孢杆菌表达系统以及杀虫菌株的构建。     

家蚕转基因载体pBacA3EG的构建及其表达

以家蚕Bombyx mori肌动蛋白A3(actin 3)启动子、增强性绿色荧光蛋白(enhanced green fluorescent protein, EGFP)基因及SV40的多聚腺苷酸识别序列为元件,经多次克隆,将其插入到piggyBac转座载体中。经PCR、酶切鉴定及测序表明各元件已按正确的方式插入到piggyBac载体中。将构建好的piggyBac表达载体显微注射到胚盘形成前期的蚕卵中,在胚胎早期发育的第3天,通过体视荧光显微镜检测到蚕卵内发出较强的绿色荧光。结果表明该载体构建正确且能在蚕卵中进行表达。家蚕转基因载体的体外瞬时表达不但是成功进行家蚕转基因所必需的第一步,而且其自身也可以应用于基因的功能研究,为家蚕后基因组研究奠定了基础。     

Expression of the hIGF-I gene driven by the Fhx/P25 promoter in the silk glands of germline silkworm and transformed BmN cells

The expression of the human insulin-like growth factor (hIGF-I) gene driven by the Fhx/P25 promoter in the silk glands of transgenic silkworms (Bombyx mori) and in transformed silkworm cells, was achieved using BmN cells transfected with a piggyBac vector, pigA3GFP-Fhx/P25-hIGF-ie-neo containing a neomycin-resistance gene (neo), a green fluorescent protein gene (gfp), an hIGF-I gene, and a helper plasmid containing the piggyBac transposase sequence under the control of the B. mori actin 3 (A3) promoter. We selected stably transformed BmN cells expressing hIGF-I using the antibiotic G418. The expression level of hIGF-I was about 450 pg in 3 × 10(6) cells, determined by ELISA. The piggyBac vector was transferred into the silkworm eggs using sperm-mediated gene transfer. The expression level of hIGF-I per gram fresh posterior silk glands of G4 transgenic silkworms was approx. 150 ng.