A novel T7 system utilizing mRNA coding for T7 RNA polymerase

The T7 system dose not require the relocation of a reporter gene to the nucleus for its gene expression in the cytoplasm, but relies on the co-localization of T7 RNA polymerase (T7 RNAP) enzyme and reporter gene DNA that is controlled by the T7 promoter. In the present study, we developed a new T7 system in that gene expression can occur at a higher level than those using conventional systems. Insertion of 5'- and 3'-untranslated regions (UTR) of beta-globin gene into a reporter gene enhanced the reporter gene expression, presumably due to the stability and efficient translation of the mRNA. Instead of the T7 RNAP protein used in conventional methods, moreover, transfection of cells with T7 RNAP mRNA, which has been modified by inserting beta-globin 5'- and 3'-UTR sequences as well as the cap and poly(A) tail structures, further enhanced the reporter gene expression. Thus, this novel T7 system using T7 RNAP mRNA may be powerful for the efficient gene expression of DNA exogenously provided in the cytoplasm.     

Cytoplasmic expression of ribozyme in zebrafish using a T7 autogene system

A cytoplasmic ribozyme expression system, based on codelivery of a ribozyme vector, a T7 autogene vector, and T7 RNA polymerase (RNAP), has been developed and used to generate a specific phenotype in zebrafish by targeting a no tail (ntl) mRNA. The expression of the no tail ribozyme sequence is under the control of a tandem of two promoters: The T7 promoter and an adenoviral va 1 (pol III) promoter. The coinjection of the ribozyme vector pT7vaRz, the T7 autogene vector pT7T7, and the T7 RNAP resulted in rapid synthesis of the ribozyme against the ntl mRNA in the cytoplasm of the injected zebrafish embryos, generating no tail phenotypes in up to 10-20% of the injected embryos. The phenotypic change rates have been found to be related to the concentrations of the plasmid vectors and T7 RNAP injected and to the ratios of the three injected components. This cytoplasmic ribozyme expression system may be useful for efficiently targeting other mRNA and for various biomedical applications. These potential applications may include rapid identification of biological functions of novel genes from zebrafish and humans based on partial gene sequence information and gene therapy of genetic and acquired diseases.     

Development of orthogonal T7 expression system in Klebsiella pneumoniae

Most expression systems are tailored for model organisms rather than nonmodel organisms. However, heterologous gene expression in model organisms constrains the innate advantages of original strain carrying gene of interest. In this study, T7 expression system was developed in nonmodel bacterium Klebsiella pneumoniae for production of chemicals. First, we engineered a recombinant K. pneumoniae strain harboring two vectors. One vector was used to express T7 RNA polymerase (T7 RNAP) which would drive the expression of egfp in the other vector. This recombinant strain demonstrated 15.73-fold of fluorescence relative to wild-type K. pneumoniae and showed similar level of fluorescence to recombinant Escherichia coli overexpressing egfp. When egfp was replaced by puuC, an endogenous aldehyde dehydrogenase catalyzing 3-hydroxypropionic acid (3-HP) biosynthesis in K. pneumoniae, the recombinant strain coexpressing T7 RNAP and PuuC showed high-level PuuC expression. In shake-flask cultivation, this recombinant strain produced 1.72 g/L 3-HP in 24 hr, which was 3.24 times that of wild-type K. pneumoniae (0.53 g/L). To mitigate plasmid burden, the vector expressing T7 RNAP was eliminated, but the T7 RNAP expression cassette was integrated into K. pneumoniae genome. The resulting strain harboring only PuuC expression vector produced 2.44 g/L 3-HP in 24 hr under shake-flask conditions, which was 1.46 times that of the strain harboring both T7 RNAP and PuuC expression vectors. In bioreactor cultivation, this strain generated 67.59 g/L 3-HP and did not show significantly halted growth. Overall, these results indicate that the engineered T7 expression system functioned efficiently in K. pneumoniae. This study provides a paradigm for the development of T7 expression system in prokaryotes.     

T7 RNA聚合酶表达系统的真核化及其偶联表达系统的建立

为了使T7RNA聚合酶(T7RNAP)表达系统真核化,首先,利用两种方法建立能够表达T7RNAP的真核细胞:(1)共转染表达T7RNAP的真核表达重组质粒于靶细胞;(2)利用稳定表达T7RNAP的BHK-21细胞系。然后,将FMDV内部核糖体进入位点(IRES)序列和增强型绿色荧光蛋白(EGFP)基因定向克隆进原核载体pET-40a-c( )的T7启动子下游,得到的重组质粒pIERS-EGFP-E。用该质粒分别转染上述两种细胞,在紫外显微镜下都能够观察到绿色荧光,表明真核化的T7RNAP偶联表达系统建立。并利用流式细胞仪对偶联表达水平进行了分析。这为利用该系统真核高效表达外源蛋白奠定了坚实的基础。用实验证明了FMDV5′端含有IERS具有介导非帽依赖性的翻译的功能,为以IERS为基础的双顺反子表达系统的建立及深入研究IERS与相关蛋白的功能奠定了基础。T7RNAP偶联表达体系是一种良好的外源基因表达体系。     

基于T7表达系统的洋葱伯克霍尔德菌G63脂肪酶同源高效表达

为实现对洋葱伯克霍尔脂肪酶的可控高效表达, 将目前被广泛使用的T7重组蛋白高效表达系统移植到洋葱伯克霍尔德菌(Burkholderia cepacia)G63中进行脂肪酶同源表达。首先采用PCR从大肠杆菌BL21(DE3)中得到T7 RNA 聚合酶基因(T7 RNAP)并将其克隆到致死质粒pJQ200SK上, 然后在T7 RNAP 前后各加入500 bp用于同源重组的片段, 再通过三亲本杂交把T7 RNAP整合到B. cepacia基因组上, 使T7 RNAP受到脂肪酶基因(lipA)启动子调控。接着把lipA和它的伴侣基因lipB单独或全部克隆到载体pUCPCM和pBBR22b上, 构建出pBBR22blipAB、pBBR22blipA、pUCPCMlipAB、pUCPCMlipA、pUCPCMΔlipAlipB、pUCPCMΔlipA、pUCPCMΔlipB七种表达质粒, 通过电转化将上述表达质粒转化到含T7 RNAP的B. cepacia宿主菌中, 最终得到一系列脂肪酶基因工程菌。通过摇瓶诱导发酵发现含表达质粒pUCPCMlipAB的工程菌脂肪酶酶活最高, 达到607 U/mg, 与野生菌相比酶活力提高2.8倍, 并且除含pUCPCMΔlipB的工程菌外, 其它工程菌的脂肪酶酶活均有不同程度提高。野生菌与工程菌pUCPCMlipAB的发酵液经硫酸铵沉淀, Sephadex G-75凝胶过滤纯化后, 比酶活分别为29 984 U/mg和30 875 U/mg。以上结果表明, 构建的基于T7表达系统的B. cepacia脂肪酶基因工程能有效提高脂肪酶的表达量, 同时说明分泌信号PelB和增强转录的核糖体接合位点对脂肪酶的表达有促进作用。     

Leishmania chagasi: a tetracycline-inducible cell line driven by T7 RNA polymerase

Trypanosomatid protozoa lack consensus promoters for RNA polymerase (RNAP) II. However, the artificial insertion of the T7 promoter (P(T7)) and the tetracycline repressor into Trypanosoma brucei cell lines expressing T7RNAP allows P(T7)-driven gene expression to be tetracycline-inducible. These cell lines provide a molecular tool to address protein function by several recombinant approaches. We describe here the development of an analogous Leishmania chagasi cell line bearing the genes for exogenous T7RNAP and the tetracycline repressor inserted in the multi-gene alpha-tubulin locus. A plasmid construct with P(T7) and the tetracycline operator upstream of a reporter gene, when introduced into this cell line as episomal plasmids or chromosomal insertion into the non-coding strand of an 18SrRNA gene, resulted in tetracycline-inducible expression of the reporter as much as 16- and 150-fold, respectively. The reporter was under a much tighter control when chromosomally inserted than extra-chromosomally born. Furthermore, P(T7) augmented the reporter's expression 2-fold more in comparison to P(T7)-less constructs. This cell line is the first Leishmania spp. that allows the exogenous T7RNAP-driven gene expression to be tetracycline-inducible; and may provide a useful tool for addressing protein function by manipulating expression levels of Leishmania endogenous genes.     

Differential adenoassociated virus vector-driven expression of a neuropeptide Y gene in primary rat brain astroglial cultures after transfection with sendai virosomes versus LipofectinTM

The ability of Sendai virosomes or Lipofectin^TM to introduce an AAV vector into primary rat brain astroglial cultures was characterized. The pJDT95npy vector was constructed by inserting rat NPY cDNA downstream from the indigenous AAV p5, p19 and p40 promoters in pJDT95. Lipofectin^TM-mediated transfection with pJDT95npy (10 g) resulted in pronounced expression of several NPY mRNA species: p5-driven (3.3 kb), p19-driven (2.7 kb) and p40-driven (0.6, 0.8, 1.1, and 1.8 kb). Exposure to virosomally encapsulated pJDT95npy (50 or 100 ng) resulted in transient expression of some p40-driven mRNA species (0.8 and 1.8 kb). Neither method produced astroglia cells which synthesized mature NPY immunoreactivity. This demonstrates that an AAV-derived vector can drive gene expression in astroglia, that Sendai virosomes can infuse vectors into astroglia, but that the amount of DNA infused in this manner may limit long term expression.     

Epi-CHO, an episomal expression system for recombinant protein production in CHO cells

This study describes the development of a transient expression system for CHO cells based on autonomous replication and retention of transfected plasmid DNA. A transient expression system that allows extrachromosomal amplification of plasmids permits more plasmid copies to persist in the transfected cell throughout the production phase leading to a significant increase in transgene expression. The expression system, named Epi-CHO comprises (1) a CHO-K1 cell line stably transfected with the Polyomavirus (Py) large T (LT) antigen gene (PyLT) and (2) a DNA expression vector, pPyEBV encoding the Py origin (PyOri) for autonomous plasmid amplification and encoding Epstein-Barr Virus (EBV) nuclear antigen-1 (EBNA-1) and OriP for plasmid retention. The CHO-K1 cell line expressing PyLT, named CHO-T was adapted to suspension growth in serum-free media to facilitate large-scale transient transfection and recombinant gene expression. Enhanced green fluorescent protein (EGFP) and human growth hormone (hGH) were used as reporter proteins to demonstrate transgene expression and productivity. Transfection of suspension-growing CHO-T cells with the vector pPyEBV encoding hGH resulted in a final concentration of 75 mg L(-1) of hGH in culture supernatants 11 days following transfection.     

Coupling of importin beta binding peptide on plasmid DNA: transfection efficiency is increased by modification of lipoplex's physico-chemical properties

Background

Non-viral vectors for gene transfer are less immunogenic than viral vectors but also less efficient. Significant effort has focused on enhancing non-viral gene transfer efficiency by increasing nuclear import of plasmid DNA, particularly by coupling nuclear localization peptidic sequences to plasmid DNA.

Results

We have coupled a 62-aminoacid peptide derived from hSRP1α importin beta binding domain, called the IBB peptide to plasmid DNA by using the heterobifunctional linker N-(4-azido-2,3,5,6 tetrafluorobenzyl)-6-maleimidyl hexanamide (TFPAM-6). When covalently coupled to plasmid DNA, IBB peptide did not increase the efficiency of cationic lipid mediated transfection. The IBB peptide was still able to interact with its nuclear import receptor, importin β, but non-specifically. However, we observed a 20-fold increase in reporter gene expression with plasmid DNA / IBB peptide complexes under conditions of inefficient transfection. In which case, IBB was associated with plasmid DNA through self assembling ionic interaction.

Conclusions

The improvement of transfection activity was not due to an improved nuclear import of DNA, but rather by the modification of physicochemical properties of IBB peptide / plasmid complexes. IBB peptide increased lipoplex size and these larger complexes were more efficient for gene transfer.

     

构建基于杆状病毒的BV-T7杂合表达体系及利用其在哺乳动物和禽类细胞中表达eGFP基因

【目的】研究重组杆状病毒BV-T7杂合表达体系能否有效转导禽类细胞并在禽类细胞中表达外源基因(eGFP),从而构建能在禽类细胞中高效稳定表达外源基因的重组杆状病毒表达系统。【方法】本研究利用Bac-to-Bac杆状病毒表达系统,结合T7表达系统,通过对eGFP表达水平的调控来把握噬菌体T7 RNA聚合酶(T7 RNAP)的功能。利用两支重组杆状病毒,pFastBac-CMV-T7 RNAP重组杆状病毒为哺乳动物细胞启动子CMV调控的噬菌体T7 RNA聚合酶的cDNA;pFB-T7pro-IRES-GFP-T7ter重组杆状病毒为T7启动子控制的eGFP报告基因。将两支重组杆状病毒共同侵染哺乳动物OL(oligodendrocyte)细胞、鸡胚成纤维细胞和鸡胚骨骼肌细胞。【结果】两支重组杆状病毒利用T7启动子和T7 RNAP,在OL细胞、鸡胚成纤维细胞和鸡胚骨骼肌细胞中成功表达eGFP报告基因,而且未引起细胞病变,但在鸡胚原代细胞中eGFP的表达相对弱于在OL细胞中的表达。在OL细胞中重组杆状病毒对细胞的转导效率为59.5%,在鸡胚成纤维细胞和鸡胚骨骼肌细胞中转导效率分别为23.2%和33.1%。【结论】本研究构建的基于杆状病毒、T7RNA聚合酶、T7启动子(BV-T7)杂合表达体系能够在哺乳类细胞及禽类细胞中表达T7 RNAP,并利用T7RNAP继续高效而稳定地表达外源基因。这为难于体外操作的RNA病毒提供了有效的研究方法,并对新型基因工程疫苗的研制提供了一个高效而稳定的表达载体系统。     

基因治疗微链载体的构建及表达分析

基因载体的转染、表达效率低和存在安全问题是基因治疗的难点。由于传统病毒及质粒载体含有大量外源基因, 其表达有可能引发较严重免疫副反应。本课题旨在新的设计思路上开发高效安全基因治疗载体。 微链载体利用设计好的Cap序列封闭基因表达框两端, 起到防止细胞内核酸外切酶降解的作用。从pEGFP-N3质粒中分离纯化得到GFP基因作为微链载体的报告基因。将微链载体与原质粒载体(pEGFP-N3质粒)分别转染入真核细胞, 利用荧光显微镜和流式细胞仪检测并比较其转染效率。结果显示微链载体在293、CNE2、3T3、B95-8等真核细胞中的转染、表达效率较高, 并具有较小的细胞毒性。初步证实了微链载体在真核细胞中转染、表达效率及安全性等方面具有一定的优越性。     

Intracellular delivery of nucleic acid by cell-permeable hPP10 peptide

Although gene therapy offers hope against incurable diseases, nonreplicating transduction vectors remain lacking. We have previously characterized a cell-penetrating peptide hPP10 for the delivery of various cargoes; however, whether hPP10 can mediate nucleic acid delivery is still unknown. Here, examining via different ways, we demonstrate that hPP10 stably complexes with plasmid DNA (pDNA) and safely mediates nucleic acid transfection. hPP10 can mediate GFP-, dsRed-, and luciferase-expressing plasmids into cells with nearly the same efficiency as commercial transfection reagents Turbofectin or Lipofect. Furthermore, hPP10 can mediate Cre fusion protein delivery and pDNA transfection simultaneously in the Cre/loxp system in vitro. In addition, hPP10 fused with an RNA-binding domain can mediate delivery of small interfering RNA into cells to silence the reporter gene expression. Collectively, our results suggest that hPP10 is an option for nucleic acid delivery with efficiencies similar to that of commercial reagents.     

Gene transfer into adult rat spinal cord using naked plasmid DNA and ultrasound microbubbles

BACKGROUND: Although gene therapy might become a promising approach to treat spinal cord injury, the safety issue is a serious consideration in human gene therapy. Plasmid DNA transfer is safer than viral vectors, but the transfection efficiency is quite low. To overcome the problem, we applied the ultrasound microbubbles-mediated transfection method to the spinal cord in adult rats, since ultrasound microbubbles have been reported to be efficient to increase transfection efficiency in various tissues. METHODS: After exposing T9-10 spinal cord with a laminectomy, we injected a mixture of naked plasmid DNA and microbubbles into cerebrospinal fluid by lumbar puncture. Then, the T9-10 spinal cord was exposed to ultrasound. CONCLUSIONS: An ultrasound intensity of 0.4-0.5 W/cm2 significantly increased luciferase expression up to approximately 15-60-fold at the insonated level as compared to naked plasmid DNA alone. Luciferase activity could be detected at least up to 7 days after transfection, while the expression level was almost returned to undetectable level at 14 days after transfection. The transfected cells were mainly meningeal cells in the surface of insonated spinal cord. There was no obvious evidence of worsening of neurological deficits as compared to rats transfected with naked plasmid DNA alone or untransfected rats. Similarly, successful gene transfer was also achieved in the insonated T9-10 spinal cord after spinal cord injury. Overall, the present study demonstrated the feasibility of ultrasound microbubbles-mediated plasmid DNA transfer into the target level of the spinal cord.     

Non-replicating Epstein-Barr virus-based plasmids extend gene expression and can improve gene therapy in vivo

To date, no gene transfer vector has produced prolonged gene expression following a single intravenous injection and then efficiently re-expressed the delivered gene following repeated systemic injection into immunocompetent hosts. To overcome these limitations, a gene therapy regimen using non-replicating Epstein-Barr virus (EBV)-based expression plasmids was developed. One plasmid contains the FR (EBV family of repeats) sequence and the expressed gene. The other encodes Epstein-Barr nuclear antigen 1 (EBNA-1), but lacks FR. Although unable to replicate in mice, intravenous co-injection of EBV-based plasmids in cationic liposome-DNA complexes (CLDCs) substantially prolonged luciferase gene expression. The use of a two-vector system limited host exposure to the EBNA-1 gene product. Furthermore, this EBV-based vector system could be intravenously re-injected multiple times into immunocompetent mice without loss of transfection efficiency. Use of this vector system significantly improved the therapeutic efficacy of the biologically important human granulocyte colony-stimulating factor gene. Delivery of the human granulocyte colony-stimulating factor gene in EBV-based plasmids increased circulating white blood counts for at least 2 months following a single CLDC-based intravenous co-injection. Conversely, white blood counts were never elevated following injection of CLDCs lacking EBV-derived elements. Thus, this EBV-based plasmid vector system both markedly prolongs gene expression at therapeutic levels and efficiently and repeatedly re-transfects immunocompetent hosts. These properties of EBV-based plasmid vectors appear to be due, at least in part, to the documented abilities of the EBNA-1 protein both to retain FR-containing DNA intracellularly and within the nucleus and to block anti-EBNA-1 cytotoxic T cell responses.     

一种新型慢病毒载体制备体系的初步建立

为了建立新型、高产量的慢病毒载体制备体系,将构建好的主框架质粒pVECRNA、包装质粒pGAGPOL及包膜质粒pVSVG通过脂质体共转染至BHK21细胞.再用含有T7RNA聚合酶基因的重组痘苗病毒vTF-3感染细胞,培养4天后,收集培养上清.提取培养上清的RNA,进行RT-PCR反应,将培养上清进行免疫印迹鉴定,将培养上清感染正常的293T细胞、HepG2细胞、Vero细胞,荧光显微镜下观察细胞GFP的表达情况,采取3×3×3析因分析方法,优化系统产量,Real-timePCR方法测定细胞培养上清中病毒载体的拷贝数,利用流式细胞术检测病毒载体滴度.RT-PCR及p24免疫印迹结果均提示在细胞上清中存在慢病毒载体;通过荧光显微镜观察到感染组293T细胞、HepG2细胞、Vero细胞均表达绿色荧光蛋白GFP,说明此系统制备出的慢病毒载体具有感染性;系统经优化后,培养上清中慢病毒载体拷贝数达到1.1×1012/ml,培养上清原始滴度达到1.3×108tu/ml,高出目前常用制备体系产量1个数量级.上述结果表明,新型慢病毒载体制备体系已初步建立,为今后该系统的大规模应用提供客观的科学依据.     

An episomal vector for stable tetracycline-regulated gene expression.

The recently introduced tetracycline (Tc)-regulatable eukaryotic gene expression system based on the Escherichia coli Tn 10 tetracycline operon has proven to be a powerful tool for controlled expression of a variety of genes in vitro as well as in vivo . Control elements of this expression system are contained in two separate plasmid vectors. The tTA vector encodes a transactivator protein and the tetP vector contains a responsive operator-promoter element (tetP) that controls gene expression depending on tTA binding. Establishment of cell lines expressing a gene of interest under tetP control requires two subsequent rounds of transfection and clonal selection after each transfection. Here we describe a modification of this system in which the tetP element is placed in an episomal EBNA-based plasmid that can be stably maintained in primate but not in rodent cells. Using HeLa and human melanoma cells, we show that upon transient or stable transfection a reporter gene is expressed in a Tc-regulated manner similar to the original system. Thus, this expression system combines the advantages of episomal vectors, such as high efficiency of transfection and time-efficient selection of mass cultures, with tight control of gene expression provided by the Tc-regulatable system.