In vivo transfection of the mouse vas deferens

To explore the possibility that specific characteristics of the epithelium of the male tract can be modified, transfections of the mouse vas deferens have been performed using in vivo injections of cationic DNA/liposome complexes. Gene transfer was done employing the reporter genes pEGFP-C1 encoding Green Fluorescent Protein (GFP) and pCMV-nls-beta encoding the nuclear beta-Galactosidase (beta-Gal). Foreign gene expression reached a maximum of 6.8% in the epithelial cells of the vas after treatment with the nuclear beta-Gal gene construction and of 13.3% after employing the GFP gene construction. Expression of the GFP gene appeared from one week up to three months following injection, and it appeared as patches of modified cells along the epithelium. Results from immunocytochemistry and Western Blotting support the conclusion that transfection of epithelial cells was achieved. We have also transfected the vas using gene constructions that express secretory proteins--specifically, the reporter system pSEAP-control that expresses a secretory form of human placental alkaline phosphatase, and the pGFP-Ctk-37 that expresses a secretion form of GFP. In both cases, the fluids expressed from the transfected vas showed a significant increase of alkaline phosphatase activity after pSEAP transfection and the presence of GFP protein when pGFP-Ctk-37 gene construction was employed. Our results indicate that the vas can be transfected in vivo using liposomes as vectors of foreign genes and that the vas fluid contents can be modified.     

Coaxial electrohydrodynamic spraying of plasmid DNA/polyethylenimine (PEI) polyplexes for enhanced nonviral gene delivery

DNA/polyethylenimine (PEI) polyplexes are an important class of nonviral vectors. Although the conventional preparation method, bulk mixing, is straightforward, the formation of the DNA/PEI polyplexes is not well controlled. This work explores coaxial electrohydrodynamic spraying (EHDS) as a novel, alternative method to produce DNA/PEI polyplexes in a more controlled manner. Both pGFP/PEI and pSEAP/PEI polyplexes were produced by EHDS with a coaxial needle setup. The size of the polyplexes was determined using dynamic light scattering, and their ability to transfect NIH 3T3 cells was observed by using an inverted fluorescence microscope (pGFP) or quantified by measuring the activity level of alkaline phosphatase (pSEAP). At nitrogen to phosphate ratio (N/P) of 6.7, the polyplexes produced by coaxial EHDS had delivery efficiencies up to 2.6 times higher than those produced by bulk mixing. The N/P ratio and the structure of the EHDS used to make the polyplexes were crucial factors in determining the delivery efficiency. Biotechnol. Bioeng. 2010. 105: 834–841. © 2009 Wiley Periodicals, Inc.     

Optimization of transient gene expression in mammalian cells and potential for scale-up using flow electroporation

The goals of this study were to identify mammalian cell lines which could be efficiently transiently-transfected and scaled-up for protein production. The transfection efficiencies of eight cell lines (NSO, NSO-TAg, CV-1, COS-7, CHO, CHO-TAg, HEK 293, and 293-EBNA) were measured using electroporation for DNA delivery and green fluorescent protein (Evans, 1996) as the reporter gene. In addition, we have evaluated the effects of stable expression of viral proteins, cell cycle manipulation, and butyrate post-treatment in small scale experiments. The cell lines varied widely in their GFP transfection efficiencies. Stable expression of simian virus 40 large T-antigen or Epstein Barr nuclear antigen failed to significantly increase transfection efficiency above that seen in the parental lines. Aphidicolin (a DNA polymerase inhibitor), which blocked cells from S or G2/M, brought about an increase in transfection efficiency in two cell lines. The primary effect of butyrate (a histone deacetylase inhibitor) post-treatment was an increased intensity of the fluorescent signal of green fluorescent protein, as measured by flow cytometry (1.0 to 4.2-fold, depending on the cell line). The combined use of aphidicolin pretreatment followed by butyrate treatment post- electroporation yielded increases in fluorescence intensities ranging from 0.9 to 6.8-fold. Based on their high transfection efficiencies in small scale experiments, rapid growth, and ability to grow in suspension culture, CHO, CHO-TAg, and 293-EBNA were selected to assess the feasibility of using flow electroporation for large-scale transfections. Using secreted placental alkaline phosphatase as a reporter, 293-EBNA cells produced the highest protein levels in both the presence and absence of butyrate. These data indicate that flow electroporation provides an efficient method of DNA delivery into large numbers of cells for mammalian protein production. This revised version was published online in August 2006 with corrections to the Cover Date.     

Adeno-associated virus vectors transduce primary cells much less efficiently than immortalized cells.

Immortalized cell lines have been used to study infection and replication of adeno-associated virus (AAV) in culture, but primary cells presumably provide a better model for AAV behavior in animals. Here, we have evaluated the ability of AAV vectors to transduce primary and immortalized strains of human epithelial cells and fibroblasts. Two AAV vectors were used, one that transduced an alkaline phosphatase gene (AAV-LAPSN), and one that transduced a beta-galactosidase/neomycin phosphotransferase fusion gene (AAV-L beta geo). The transduction efficiency of the AAV-LAPSN vector, quantitated by measurement of alkaline phosphatase-positive cell foci following infection, was 10 to 60 times greater in immortalized human cells than in primary cells, and total alkaline phosphatase activity in cell lysates was 40 to 50 times greater in immortalized cells. The AAV-L beta geo vector gave similar results. In contrast, the transduction efficiency of a retrovirus vector encoding alkaline phosphatase was equivalent in primary and immortalized cells. Analysis of the quantity and state of the AAV vector genomes in cells showed that primary and immortalized cells contained comparable numbers of vector copies per cell and that the vast majority of vector DNA was not integrated into the cell genome. Additionally, the level of AAV vector-derived message paralleled the transduction efficiency. These results indicate that the block to functional transduction in primary cells occurred after virus entry and limited the abundance of vector-derived message. Data from AAV transduction in cultures of human cells containing immortalizing genes suggest that cellular changes secondary to the introduction of immortalizing genes increased permissiveness for transduction by AAV vectors. In summary, our data demonstrate that AAV vectors transduce primary human cells much less efficiently than immortalized cells and indicate the importance of using primary cells to evaluate AAV vectors for gene therapy applications.     

Construction of Epstein-Barr virus-based expression vector containing mini-oriP.

Epstein-Barr virus (EBV)-based vectors are extrachromosomal vectors carrying a replicational origin, oriP (about 2200 bp) and a replication initiation factor (EBNA-1) which are sufficient for autonomous replication. Because one disadvantage of these vectors is their large sizes, we examined the effect of partial deletion of oriP on the effectiveness of the EBV-based vectors, using an enhanced green fluorescent protein (EGFP) as a reporter to monitor gene expression. Results indicated that 954 bp-deleted mini-oriP is useful in primate cells since the vector showed high efficiency of stable transfection, a high ratio of EGFP-positive cells, and high recovery of intact plasmid DNA from transfected cells.     

Copy number of adenoviral vector genome transduced into target cells can be measured using quantitative PCR: application to vector titration

Both transfection and adenovirus vectors are commonly used in studies measuring gene expression. However, the real DNA copy number that is actually transduced into target cells cannot be measured using quantitative PCR because attached DNA present on the cell surface is difficult to distinguish from successfully transduced DNA. Here, we used Cre/loxP system to show that most of the transfected DNA was in fact attached to the cell surface; in contrast, most of the viral vector DNA used to infect the target cells was present inside the cells after the cells were washed according to the conventional infection protocol. We applied this characteristic to adenoviral vector titration. Current methods of vector titration using the growth of 293 cells are influenced by the effect of the expressed gene product as well as the cell conditions and culture techniques. The titration method proposed here indicates the copy numbers introduced to the target cells using a control vector that is infected in parallel (relative vector titer: rVT). Moreover, the new titration method is simple and reliable and may replace the current titration methods of viral vectors.     

High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells

A scalable transfection procedure using polyethylenimine (PEI) is described for the human embryonic kidney 293 cell line grown in suspension. Green fluorescent protein (GFP) and human placental secreted alkaline phosphatase (SEAP) were used as reporter genes to monitor transfection efficiency and productivity. Up to 75% of GFP-positive cells were obtained using linear or branched 25 kDa PEI. The 293 cell line and two genetic variants, either expressing the SV40 large T-antigen (293T) or the Epstein–Barr virus (EBV) EBNA1 protein (293E), were tested for protein expression. The highest expression level was obtained with 293E cells using the EBV oriP-containing plasmid pCEP4. We designed the pTT vector, an oriP-based vector having an improved cytomegalovirus expression cassette. Using this vector, 10- and 3-fold increases in SEAP expression was obtained in 293E cells compared with pcDNA3.1 and pCEP4 vectors, respectively. The presence of serum had a positive effect on gene transfer and expression. Transfection of suspension-growing cells was more efficient with linear PEI and was not affected by the presence of medium conditioned for 24 h. Using the pTT vector, >20 mg/l of purified His-tagged SEAP was recovered from a 3.5 l bioreactor. Intracellular proteins were also produced at levels as high as 50 mg/l, representing up to 20% of total cell proteins.     

Effects of pCIneo and pCEP4 expression vectors on transient and stable protein production in human and simian cell lines

To support and meet the demand for recombinant proteins early in the drug discovery process, much work has been directed toward improving the methods used for transient gene transfection and expression. A factor which could potentially affect the outcome of experiments is the choice of the expression vector. Conventional vectors such as pCIneo and pcDNA3 have been used frequently. Each of these places the gene of interest under the control of the CMV promoter. An interesting alternative is provided by episomal vectors. For example, the pCEP4 vector contains the gene coding for the Epstein Barr nuclear antigen as well as the EBNA ori P sequence. This combination allows for the episomal replication of the plasmid. In preliminary experiments, we compared transient secreted placental alkaline phosphatase production in 8 cell lines from 3 different species using the pCIneo vs. pCEP4 vectors and found the utility of the pCEP4 vector to be limited to the human 293 EBNA cell line. In this paper, we have compared the two vectors in six cell lines of simian and human origin, measuring the transient production of secreted placental alkaline phosphatase and human hepatocyte growth factor. In general, the pCEP4 vector produced higher amounts of both proteins in transient transfections. Results were particularly pronounced in the HEK 293 and 293 EBNA cell lines. Stable pools of cells (uncloned) expressing human hepatocyte growth factor were isolated using pCIneo and pCEP4 and protein production levels were compared to those seen in transient transfections. Stable expression with pCEP4 was found to produce the highest levels of human hepatocyte growth factor in 3 of 4 cell lines. Finally, electroporation and FuGENE^TM6(Roche, Indianapolis IN) as transfection methods were compared measuring transient production of secreted placental alkaline phosphatase, human hepatocyte growth factor, and green fluorescent protein. FuGENE produced higher protein concentrations in less time than electroporation for all 3 proteins. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transient production of recombinant proteins by Chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents

We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG4 encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 microg mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn297 was not significantly affected by nocodazole during transient production by this method.     

Design and packaging of adeno-associated virus gene targeting vectors

Adeno-associated virus (AAV) vectors can transduce cells by several mechanisms, including (i) gene addition by chromosomal integration or episomal transgene expression or (ii) gene targeting by modification of homologous chromosomal sequences. The latter process can be used to correct a variety of mutations in chromosomal genes with high fidelity and specificity. In this study, we used retroviral vectors to introduce mutant alkaline phosphatase reporter genes into normal human cells and subsequently corrected these mutations with AAV gene targeting vectors. We find that increasing the length of homology between the AAV vector and the target locus improves gene correction rates, as does positioning the mutation to be corrected in the center of the AAV vector genome. AAV-mediated gene targeting increases with time and multiplicity of infection, similar to AAV-mediated gene addition. However, in contrast to gene addition, genotoxic stress did not affect gene targeting rates, suggesting that different cellular factors are involved. In the course of these studies, we found that (i) vector genomes less than half of wild-type size could be packaged as monomers or dimers and (ii) packaged dimers consist of inverted repeats with covalently closed hairpins at either end. These studies should prove helpful in designing AAV gene targeting vectors for basic research or gene therapy.     

Efficient mouse airway transduction following recombination between AAV vectors carrying parts of a larger gene

The small packaging capacity of adeno-associated virus (AAV) vectors limits the utility of this promising vector system for transfer of large genes. We explored the possibility that larger genes could be reconstituted following homologous recombination between AAV vectors carrying overlapping gene fragments. An alkaline phosphatase (AP) gene was split between two such AAV vectors (rec vectors) and packaged using AAV2 or AAV6 capsid proteins. Rec vectors having either capsid protein recombined to express AP in cultured cells at about 1-2% of the rate observed for an intact vector. Surprisingly, the AAV6 rec vectors transduced lung cells in mice almost as efficiently as did an intact vector, with 10% of airway epithelial cells, the target for treatment of cystic fibrosis (CF), being positive. Thus AAV rec vectors may be useful for diseases such as CF that require transfer of large genes.     

Selectable plasmid vectors with alternative and ultrasensitive histochemical marker genes

Three different histochemical marker genes--E. coli beta-galactosidase gene (lacZ), Drosophila alcohol dehydrogenase gene (ADH) and human placenta alkaline phosphatase gene (ALP)--were cloned into a eukaryotic expression vector also containing the neomycin resistance gene. After calcium phosphate transfection and G418 sulfate selection of recipient BALB/c 3T3 cells, stable transfectants were pooled for histochemical staining. The lacZ-bearing cells produce aqua blue staining for beta-galactosidase; ADH-bearing cells, blue-black staining for alcohol dehydrogenase; and ALP-bearing cells, red staining for alkaline phosphatase. Cells carrying different marker genes can be easily differentiated by double-staining protocols. In addition, various photographic films can be used to enhance the colors of specific histochemically tagged cell classes. These plasmid vectors, providing selectability with the neomycin resistance gene and ultrasensitivity of alternative histochemical marker genes, will be very effective in virtually any biological system requiring analyses of multiple cell clones or classes in culture model systems or in situ.     

Co-transfection with the osteogenic protein (OP)-1 gene and the insulin-like growth factor (IGF)-I gene enhanced osteoblastic cell differentiation

Previous studies from this laboratory showed that the action of Osteogenic Protein-1 (OP-1, BMP-7) on osteoblastic cell differentiation could be enhanced by other protein factors, such as Insulin-like Growth Factor (IGF)-I. In the present study, we examined the effects of co-transfection with a combination of the OP-1 and the IGF-I gene on osteoblastic cell differentiation. The results first showed that fetal rat calvaria (FRC) cells transfected with the OP-1 gene under the control of the cytomegalovirus (CMV) promoter showed substantial production of the OP-1 protein. Transfected FRC cells also showed a DNA concentration-dependent increase in alkaline phosphatase (AP) activity, an osteoblastic cell differentiation marker. Von Kossa-positive nodules, a hallmark of bone formation in long-term cultures of bone-derived cells, were also observed in the transfected cells after 26 days in culture, whereas none were observed in control cells. Co-transfection of FRC cells with the combination of the OP-1 and the IGF-I gene resulted in a synergistic stimulation of AP activity. The increase was DNA dose-dependent. The current data show that transfection of OP-1 gene into osteoblastic cells stimulates osteoblastic cell differentiation in vitro. The study further demonstrates the feasibility of employing gene transfer of a second gene in combination with an OP-1 vector to synergistically enhance OP-1 activity.     

A severe de novo methylation of episomal vectors by human ES cells

Episomal vectors can allow efficient genetic modification of cells and have the potential advantage of avoiding chromosomal position of integration effects. Here we explore the use of an Epstein-Barr virus-based episomal vector with human embryonic stem (ES) cells, and find high initial transfection rates, but a rapid loss of reporter gene expression. Similar to mouse ES cells, human ES cells express high levels of the de novo DNA methyltransferases, and we detected dramatic CpG methylation and minor non-CpG methylation on the episomes recovered from the human ES cells 7 days after the transfection, which was not present on the same episome recovered from 293 cells. Interestingly, the oriP region of the episomes was relatively excluded from this methylation. These findings define some of the limitations of using episomal vectors with human ES cells and offer a unique platform for analyzing epigenetic gene silencing in human ES cells.     

Improved transfection efficiency of cultured human cells

We simultaneously tested the transfection efficiency of NT2/D1 and HeLa cells with Lipofectamine (Life Technologies) and Effectene (Qiagen) transfection reagents using the pCH110 eukaryotic assay vector, which contains the lacZ reporter gene. Under our culture conditions for NT2/D1 and HeLa cells, Effectene transfection efficiency could be augmented by simply increasing the amount of plasmid DNA 1.5-3 times above the recommended concentration without any visible cytotoxicity. With the Lipofectamine reagent, optimal transfection efficiency was obtained for both cell lines within the recommended concentrations, but at the top of the range. The results indicate that optimization of the transfection process should include plasmid DNA concentrations above the levels suggested by the manufacturers, in order to accomplish the highest transfection efficiency.