Introduction of the human growth hormone gene into the guinea pig mammary gland by in vivo transfection promotes sustained expression of human growth hormone in the milk throughout lactation

We tested the feasibility of transfecting mammary tissue in vivo with an expression plasmid encoding the human growth hormone (hGH) gene, under the control of the cytomegalovirus promoter. Guinea pig mammary glands were transfected with plasmid DNA infused through the nipple canal and expression was monitored in control and transfected glands by radioimmunoassay of milk samples for hGH. Sustained expression of hGH throughout lactation was attained with a polyion transfection complex shown to be optimal for the transfection of bovine mammary cells, in vitro. However, contrary to expectations, hGH expression was consistently 5- to 10-fold higher when DEAE-dextran was used alone for transfection. Thus polyion complexes which are optimal for the transfection of cells in vitro may not be optimal in vivo. The highest concentrations of hGH in milk were obtained when glands were transfected within 3 days before parturition. This method may have application for studying the biological role or physical properties of recombinant proteins expressed in low quantities, or for investigating the regulation of gene promoters without the need to construct viral vectors or produce transgenic animals.     

Human growth hormone as a reporter gene in regulation studies employing transient gene expression.

The human growth hormone (hGH) transient assay system described here is based on the expression of hGH directed by cells transfected with hGH fusion genes. Levels of secreted hGH in the medium, measured by a simple radioimmunoassay, are proportional to both levels of cytoplasmic hGH mRNA and the amount of transfected DNA. The system is extremely sensitive, easy to perform, and is qualitatively different from other transient expression systems in that the medium is assayed and the cells themselves are not destroyed. The hGH transient assay system is appropriate for analyses of regulation of gene expression and was utilized here to investigate the effect of the simian virus 40 enhancer on the herpes simplex virus thymidine kinase promoter and the effect of zinc on the mouse metallothionein-I promoter. The expression of hGH can also be used as an internal control to monitor transfection efficiency along with any other transient expression system. All cell types tested thus far (including AtT-20, CV-1, GC, GH4, JEG, L, and primary pituitary cells) were able to secrete hGH into the medium.     

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.     

用Bac-to-Bac杆状病毒系统表达人生长激素

利用Bac to Bac杆状病毒载体表达系统将人生长激素 (humangrowthhormone ,hGH)基因cDNA克隆至转移载体pFastBac1中 ,得到pFastBac hGH ,再将其转化进入含穿梭载体Bacmid的受体菌DH10Bac中 ,发生转座作用 ,得到含hGH基因的重组穿梭载体rBacmid hGH .纯化DNA ,直接转染培养的昆虫细胞Sf9,得到重组病毒rAcV Bac hGH .经酶切PCR及Southern杂交鉴定 ,hGH基因正确地插入病毒基因组的多角体蛋白基因启动子下 ,SDS PAGE测得产物蛋白分子量为 2 2kD左右 .用免疫化学发光法测得转染上清中hGH表达水平可达 18μg ml ,与用传统的BEVS表达hGH相比 ,转染上清中hGH表达水平提高 4 0 0倍以上     

人生长激素慢病毒载体的构建及其在鼠骨骼肌成肌细胞中的表达

构建能表达人生长激素(hGH)的pLentivirus6/V5-hGH载体,并实现hGH基因在骨骼肌成肌细胞中大量、长期和稳定的表达。体外培养SD鼠骨骼肌成肌细胞,并通过免疫组织化学方法鉴定所得细胞、用台酚兰染色确定培养细胞的活性并绘制生长曲线。将目的基因hGH亚克隆到真核细胞表达载体pLenti6/V5-D-TOPO载体上,构建重组质粒pLentivirus6/V5-hGH。将pLenti6/V5-hGH及阳性对照质粒pLenti6/V5-EGFP分别用Lipofectamin2000介导转染体外培养的SD乳鼠骨骼肌成肌细胞。在激光共聚焦扫描显微镜下计数,确定阳性对照质粒的转染数,从而估计该基因的转染效率。加入筛选试剂以获得稳定表达异源生长激素(GH)的成肌细胞。收集转染及筛选后的细胞培养基,用放射免疫分析法(RIA)检测重组人生长激素(rhGH)的表达水平。聚合酶链式反应法(PCR)及DNA测序显示hGH基因成功地插入到pLenti6/V5-D-TOPO载体中;阳性对照质粒转染细胞24h后,在激光共聚焦显微镜下观察,其转染效率达40%以上。检测收集的上清,与对照组相比,有极显著差异(P<0.01),观察至第8周,rhGH仍持续稳定表达。通过检测培养的chang-liver肝细胞上清中胰岛素样生长因子-1(IGF-1)的水平,验证了rhGH的生物学活性。实验通过培养高纯度的成肌细胞,构建了能在真核细胞内表达hGH的重组质粒pLenti6/V5-hGH,实现了hGH基因在骨骼肌成肌细胞中大量、长期和稳定的表达,并且获得的rhGH具有较强的促进肝细胞分泌IGF-1的能力。     

Synaptogyrins regulate Ca2+-dependent exocytosis in PC12 cells.

Synaptogyrins constitute a family of synaptic vesicle proteins of unknown function. With the full-length structure of a new brain synaptogyrin isoform, we now show that the synaptogyrin family in vertebrates includes two neuronal and one ubiquitous isoform. All of these synaptogyrins are composed of a short conserved N-terminal cytoplasmic sequence, four homologous transmembrane regions, and a variable cytoplasmic C-terminal tail that is tyrosine-phosphorylated. The localization, abundance, and conservation of synaptogyrins suggest a function in exocytosis. To test this, we employed a secretion assay in PC12 cells expressing transfected human growth hormone (hGH) as a reporter protein. When Ca2+-dependent hGH secretion from PC12 cells was triggered by high K+ or alpha-latrotoxin, co-transfection of all synaptogyrins with hGH inhibited hGH exocytosis as strongly as co-transfection of tetanus toxin light chain. Synaptophysin I, which is distantly related to synaptogyrins, was also inhibitory but less active. Inhibition was independent of the amount of hGH expressed but correlated with the amount of synaptogyrin transfected. Inhibition of exocytosis was not observed with several other synaptic proteins, suggesting specificity. Analysis of the regions of synaptogyrin required for inhibition revealed that the conserved N-terminal domain of synaptogyrin is essential for inhibition, whereas the long C-terminal cytoplasmic tail is largely dispensable. Our results suggest that synaptogyrins are conserved components of the exocytotic apparatus, which function as regulators of Ca2+-dependent exocytosis.     

Selective packaging of human growth hormone into synaptic vesicles in a rat neuronal (PC12) cell line

We have introduced the gene for human growth hormone (hGH) into PC12 cells, a rat pheochromocytoma-derived cell line with neuronal characteristics, and have isolated stable cell lines that express this protein. hGH is stored within the cells in membrane-bounded vesicles that are indistinguishable from the endogenous catecholaminergic synaptic vesicles. When the transfected cells are stimulated by carbachol or direct depolarization, they release norepinephrine and hGH with parallel kinetics. Treatment of the transfected cells with nerve growth factor results in a twofold increase in the amounts of hGH stored in and secreted from the cells. Not all proteins are packaged into the synaptic vesicles, since the rate of release of laminin, a soluble secreted protein endogenous to PC12 cells, is not stimulated by carbachol. This neuronal cell line therefore possesses at least two distinct pathways for secretion and can selectively package a foreign endocrine hormone into the regulated pathway.     

Recombinant gene expression in pulmonary vascular endothelial cells: polarized secretion in vivo

A unique, spontaneously immortalized rat pulmonary endothelial cell line was transfected with a human growth hormone (hGH) fusion gene generating a line of stably transfected cells that expresses high levels of hGH (Ec/XGH-1). These cells produced significant serum levels of hGH when implanted intraperitoneally, subcutaneously, or intravenously into nude mice. Implantation of Ec/XGH-1 cells under the renal capsule resulted in the formation of large cysts that contained concentrations of hGH that were several thousand times greater than those in serum assayed simultaneously from the same animals. This study presents a new technique for in vivo gene expression using a convenient line of pulmonary vascular endothelial cells as gene carriers. In addition, this system demonstrates the special physiological features of transfected endothelial cells in forming large cysts.     

A cell-based reporter assay for the identification of protein kinase C activators and inhibitors

Transmission of extra cellular signals across biological membranes results in the generation of lipid metabolites which in turn influence specific cellular events such as cell growth or differentiation. Many of these lipid messengers can activate protein kinase C (PKC) isozymes of which one function is to perpetuate the extracellular signals to the nucleus by phosphorylating other targets proteins. We have engineered mammalian cell lines to identify and evaluate activators and inhibitors of PKC-dependent and independent signal transduction pathways. The A31 mouse fibroblast cell line, has been stably transfected with a construct containing a triplet repeat of the TPA response element (TRE) upstream of a thymidine kinase promoter fused to the human growth hormone (hGH) gene. A31 cells containing this reporter construct exhibit significant increases in hGH secretion following stimulation by phorbol esters or other mitogens. The levels of hGH secretion are modulated in this system using different pharmacological agents. We demonstrate that this assay can be used to identify specific and general inhibitors as well as activators of the signal transduction pathway mediated by PKC isozymes. (Mol Cell Biochem141: 129–134, 1994)     

Sustained high-yield production of recombinant proteins in transiently transfected COS-7 cells grown on trimethylamine-coated (hillex) microcarrier beads

The present study shows that COS-7 cells transiently transfected and maintained on positively charged (trimethylamine-coated) microcarrier beads synthesize recombinant protein at higher levels and for longer periods of time than cells transfected and maintained on polystyrene flasks in monolayer culture. Sustained, high-level synthesis was observed with secreted chimeric proteins (murine E-selectin- and P-selectin-human IgM chimeras) and a secreted hematopoietic growth factor (granulocyte-macrophage colony-stimulating factor). Studies with green fluorescent protein indicated that the transfected cells attached more firmly to the trimethylamine-coated microcarriers than to polystyrene flasks. After 10-14 days in culture, most of the transfected cells detached from the surface of the polystyrene flasks, whereas most transfected cells remained attached to the microcarriers. The transiently transfected microcarrier cultures produced higher levels of protein per transfected cell due to this prolonged attachment. The prolonged attachment and higher output of transfected cells on microcarriers resulted in a 5-fold increase in protein production from a single transfection over two weeks. Thus, microcarrier-based transient transfection yields quantities of recombinant proteins with a significant savings of time and reagents over monolayer culture.     

High-level, heat-regulated synthesis of proteins in eukaryotic cells

Plasmids have been constructed in which promoters of 70-kDa heat-shock protein genes (hsp70) of human and Drosophila origin were linked to three different eukaryotic genes encoding human growth hormone (hGH), chicken lysozyme (cL) and a human influenza haemagglutinin (HA). Following transfection into widely divergent eukaryotic cells, the hybrid genes direct the transient, heat-regulated synthesis of the three proteins. hGH and cL are secreted into the medium. A human hsp70-hGH construct was used to establish stable mouse fibroblast lines that are capable of producing and secreting hGH at high levels following heat induction: hGH is secreted at a 500-1200-fold higher rate by heat-treated than by untreated cells.     

The use of chimeric gene constructs to express a bacterial endoglucanase in mammalian cells.

The synthesis and secretion of a truncated Clostridium thermocellum endoglucanase (EGE') encoded by the celE' gene was investigated in Chinese hamster ovary (CHO) cells. Fusion genes consisting of the human growth hormone (hGH) gene and celE', transcribed from the SV40 early enhancer/promoter, were constructed and stably transfected into CHO cells. A gene consisting of celE' inserted into the first exon of the hGH gene resulted in the synthesis of truncated proteins (less than or equal to 22 kDa) lacking endoglucanase activity. Cloning celE' into the second exon of the hGH gene, resulted in the synthesis and secretion of a 50 kDa protein with endoglucanase activity. A 50 kDa protein was also synthesised by cells transfected with celE' cloned into the fifth exon of the hGH gene. However, despite a 5-fold increase in enzyme activity compared to the exon 2 transfected cell line less than 40% of the protein was secreted. Constructs devoid of introns, in which celE' was fused to the SV40 early promoter and to the rabbit beta-globin polyadenylation sequence resulted in a 2-18-fold increase in endoglucanase activity compared to the constructs containing introns. In addition more than 75% of the synthesised protein was secreted. Analyses of EGE' encoded mRNA from the transfected cell lines suggests that the presence of introns results in the aberrant splicing of message by the use of cryptic splice sites in the celE' gene. These results demonstrate that introns are not required for the efficient expression of a bacterial endoglucanase in mammalian cells, rather introns appear to reduce expression of the encoded protein.     

Non-viral transfection of goat germline stem cells by nucleofection results in production of transgenic sperm after germ cell transplantation

Germline stem cells (GSCs) can be used for large animal transgenesis, in which GSCs that are genetically manipulated in vitro are transplanted into a recipient testis to generate donor‐derived transgenic sperm. The objectives of this study were to explore a non‐viral approach for transgene delivery into goat GSCs and to investigate the efficiency of nucleofection in producing transgenic sperm. Four recipient goats received fractionated irradiation at 8 weeks of age to deplete endogenous GSCs. Germ cell transplantations were performed 8–9 weeks post‐irradiation. Donor cells were collected from testes of 9‐week‐old goats, enriched for GSCs by Staput velocity sedimentation, and transfected by nucleofection with a transgene construct harboring the human growth hormone gene under the control of the goat beta‐casein promoter (GBC) and a chicken beta‐globin insulator (CBGI) sequence upstream of the promoter. For each recipient, transfected cells from 10 nucleofection reactions were pooled, mixed with non‐transfected cells to a total of 1.5 × 10⁸ cells in 3 ml, and transplanted into one testis (n = 4 recipients) by ultrasound‐guided cannulation of the rete testis. The second testis of each recipient was removed. Semen was collected, starting at 9 months after transplantation, for a period of over a year (a total of 62 ejaculates from four recipients). Nested genomic PCR for hGH and CBGI sequences demonstrated that 31.3% ± 12.6% of ejaculates were positive for both hGH and CBGI. This study provides proof‐of‐concept that non‐viral transfection (nucleofection) of primary goat germ cells followed by germ cell transplantation results in transgene transmission to sperm in recipient goats. Mol. Reprod. Dev. 79: 255–261, 2012. © 2011 Wiley Periodicals, Inc.     

Bcl-2 small hairpin RNAs enhance radiation-induced apoptosis in A549 cells

Bcl-2, a prominent member of the family of proteins, is responsible for dys-regulation of apoptosis and resistance to chemotherapy and radiotherapy. This study investigated whether small hairpin RNA (shRNA) targeting Bcl-2 could render A549 cells more susceptible to gamma radiation-induced apoptosis. Recombinant Bcl-2 shRNAs expression vector were transfected into A549 cells with Lipofectamine 2000. Transfected cells were screened in 800 mg/ml G418 screening medium, and after stable transfection, silencing was examined. Expression of the Bcl-2 protein was assayed using Western blot in A549 cells. Inhibition of cell growth was assessed by a MTT assay. Apoptosis was determined by morphological observation and flow cytometry. Expression levels of Bcl-2 protein from A549 cells decreased after stable transfection with Bcl-2 shRNAs. No differences in Bcl-2 protein levels between control shRNA group and untreated cells were noted. After stable transfection with Bcl-2 shRNAs the viability of cells was less than after stable transfection with those with control shRNAs and untransfected A549, respectively (P<0.05). Control shRNA had no significant effect on growth of cells. Radiation significantly inhibited the growth of cells stably transfected with Bcl-2 shRNA (P<0.05). No difference in survival between the cells with control shRNA and untransfected cells was noted. Using Giemsa staining, cells stably transfected with Bcl-2 shRNA combined with radiation at 48 h displayed changes of apoptosis. After treatment with radiation apoptotic rates of the A549 cells stably transfected with Bcl-2 shRNA significantly increased (P<0.05), compared with the cells with control shRNA and untransfected cells. shRNAs against the Bcl-2 mRNA increases radiation-induced apoptosis in A549 cells.     

The quantitation of human growth hormone by a radioreceptor assay using an established human cell line

Membrane receptors on cultured human lymphocytes (IM-9) have been shown to bind human growth hormone (hGH) in a specific manner. The aim of the present study was to develop an in vitro assay of hGH based on this binding. The assay should fulfil established pharmacopoeial requirements for quantitation of hormones. The binding of [125I]hGH was studied as a function of time, temperature, cell density, tracer concentration and the concentration of unlabelled hGH and other related hormones. Also, the dissociation of bound hGH and the chemical stability of hGH in the incubation medium were studied. From these studies, the conditions for an appropriate radioreceptor assay were determined. Briefly, 1.5-3.0 X 10(7) cells ml-1 were incubated with 5-20 X 10(-12) M [125I]hGH and three different concentrations of unlabelled hGH chosen from the linear part of the [125I]hGH displacement curve. The results were analyzed according to general pharmacopoeial principles. The mean values for growth hormone activity tested by radioreceptor assay were within the fiducial limits (P = 0.05) of the corresponding activity determined by the hypophysectomized rat body-weight gain assay. The in vitro assay was found to be more precise and less resource demanding than the in vivo bioassay of hGH. It is concluded that the in vitro bioassay described here is well suited as a screening method for potency determination of hGH preparations.     

High-level expression of the Epstein-Barr virus EBNA1 protein in CV1 cells and human lymphoid cells using a SV40 late replacement vector

To construct a recombinant plasmid designed to yield large amounts of the Epstein-Barr virus (EBV) nuclear antigen, EBNA1, the EBV BamHI-K fragment (B95-8 strain) was inserted into an expression vector composed of SV40 and pBR322 DNA. The vector replicates in both Escherichia coli and eukaryotic cells. Introduction of such a BamHI-K-containing vector into CV1 monkey cells (using DEAE-dextran, glycerol and chloroquine diphosphate) gave high yields of the correct size EBNA1 protein in 40-50% of the transfected cells. Maximal amounts of EBNA1 could be extracted from the cells at 65-72 h post transfection. Using a quantitative ELISA assay, it was estimated that transfected cells express 500-1000 times more EBNA1 than lymphoid cells, latently infected with EBV. A monoclonal antibody directed against EBNA1 immunoprecipitated two proteins of 74 and 62 kDa from transfected cells. These same two proteins were detected in immunoprecipitation and immunoblot experiments using human EBV-positive polyclonal serum, although this serum also detected several other protein products in transfected cells. In vivo labelling of transfected cells with [32P]orthophosphate showed that the 74- and 62-kDa proteins are modified by phosphorylation. The same vector construction was also used to transfect an EBV-negative human lymphoblastoid cell line (Ramos). Expression of the EBNA1 protein was obtained in up to 20% of the cells.     

CD47基因对食道癌细胞增殖的影响

本研究将CD47-si RNA转染至食道癌细胞,采用蛋白免疫印迹检测食道癌细胞中CD47蛋白的表达,MTT法检测CD47-siRNA转染组和空质粒转染组(对照组)食道癌细胞增殖状态,蛋白免疫印迹检测CD47-siRNA转染组和空质粒转染组(对照组)食道癌细胞中PCNA蛋白表达,DCFDA染色流式细胞仪检测食道癌细胞CD47-siRNA转染组和空质粒转染组(对照组)中ROS水平,以探究CD47基因对食道癌发生发展的影响。研究结果表明,CD47-si RNA转染组食道癌细胞中CD47蛋白明显低于对照组;CD47-siRNA转染组食道癌细胞增殖率显著低于对照组(p<0.05);CD47-siRNA转染组细胞增殖相关蛋白PCNA低于对照组(p<0.01);CD47-siRNA转染组食道癌细胞中ROS水平明显高于对照组(p<0.05)。本研究初步认为:CD47-siRNA可降低食道癌细胞中CD47蛋白表达,抑制食道癌细胞的增殖并增加食道癌细胞中ROS水平。