Targeting vector configuration and method of gene transfer influence targeted correction of theAPRT gene in Chinese hamster ovary cells

A 21-bp deletion in the third exon of theAPRT gene in Chinese hamster ovary (CHO) cells was corrected by transfection with a plasmid containing hamsterAPRT sequences. Targeted correction frequencies in the range of 0.3–3.0×10^–6 were obtained with a vector containing 3.2 kb ofAPRT sequence homology. To examine the influence of vector configuration on targeted gene correction, a double-strand break was introduced at one of two positions in the vector prior to transfection by calcium phosphate-DNA coprecipitation or electroporation. A double-strand break in the region ofAPRT homology contained in the vector produced an insertion-type vector, while placement of the break just outside the region of homology produced a replacement-type vector. Gene targeting with both linear vector configurations yielded equivalent ratios of targeted recombinants to nontargeted vector integrants; however, targeting with the two different vector configurations resulted in different distributions of targeted recombination products. Analysis of 66 independent APRT⁺ recombinant clones by Southern hybridization showed that targeting with the vector in a replacement-type configuration yielded fewer targeted integrants and more target gene convertants than did the integration vector configuration. Targeted recombination was about fivefold more efficient with electroporation than with calcium phosphate-DNA coprecipitation; however, both gene transfer methods produced similar distributions of targeted recombinants, which depended only on targeting vector configuration. Our results demonstrate that insertion-type and replacement-type gene targeting vectors produce similar overall targeting frequencies in gene correction experiments, but that vector configuration can significantly influence the yield of particular recombinant types.     

Gene transfection of human mesenchymal stem cells with a nano‐hydroxyapatite–collagen scaffold containing DNA‐functionalized calcium phosphate nanoparticles

This study aimed to fabricate a growth factor‐releasing biodegradable scaffold for tissue regeneration. We prepared multishell calcium phosphate (CaP) nanoparticles functionalized with DNA, polyethyleneimine (PEI), protamine and octa‐arginine (R8) and compared their respective transfection activity and cell viability measures using human mesenchymal stem cells. DNA–protamine complexes improved the transfection efficiency of CaP nanoparticles with the exception of those functionalized with R8. These complexes also greatly reduced the cytotoxicity of PEI. In addition, we also fabricated DNA–protamine‐functionalized CaP nanoparticle‐loaded nano‐hydroxyapatite–collagen scaffolds and investigated their gene transfection efficiencies. These experiments showed that the scaffolds were associated with moderate hMSC cell viability and were capable of releasing the BMP‐2 protein into hMSCs following gene transfection. In particular, the scaffold loaded with protamine‐containing CaP nanoparticles showed the highest cell viability and transfection efficiency in hMSCs; thus, it might be suitable to serve as an efficient growth factor‐releasing scaffold.     

Gene transfer by adenovirus-mimetic peptides in the presence of a cationic lipid and/or adenovirus. Analysis of the contribution of the viral and nonviral components

Summary.  Peptide and cationic lipid-based gene transfer vectors have shown promise for gene therapy but are still less efficient than viral gene transfer vectors. We have examined the mechanism of gene transfer of different adenovirus-mimetic peptides in the presence and absence of a cationic lipid, lipofectamine and/or adenovirus with the aim of improving the design of nonviral vectors for efficient gene transfer. Three polylysine-adenovirus-mimetic peptides were synthesised and examined for their efficacy for gene transfer. Transfection levels in four cell lines: adenovirus permissive human tracheal epithelial (56FHTE8o⁻), human lung carcinoma (A549), human colon carcinoma (Caco-2) cells, and adenovirus low-permissive Chinese hamster ovary (CHO) cells, were examined. The polylysine-adenovirus-mimetic peptides increased the level of transfection of a reporter transgene in all cell lines. Transfection was substantially increased when an adenovirus was added to cells after pre-incubation with the vector complexes. Formulation of the peptide vector complexes with lipofectamine increased their transfection efficacy and the subsequent addition of an adenovirus increased transfection levels even further but only in permissive cells. Pre-incubation of cells with lipofectamine-peptide vector complexes increased cell binding of the adenovirus but uptake was only increased in intermediate- or non-permissive cells. The addition of lipofectamine increased transgene expression of a recombinant adenovirus in non-permissive cells but not in permissive cells. Enhancement with an adenovirus of peptide vector gene transfer is probably due to more efficient endosome escape while enhancement of gene transfer by peptide vectors complexed to lipofectamine is due to an increase in cellular binding and/or internalisation of the adenovirus. Received February 8, 2002; accepted August 23, 2002     

Optimization of gene transfer in cultured insect cells

Summary The use of polybrene for the efficient transfection of cultured mosquito cells is described. Cells are transfected with purified plasmid DNA coding for a bacterial chloramphenicol acetyltransferase (CAT) gene regulated by sequences from aDrosophila heat shock protein promoter. The DNA is added to the cells in serumfree medium containing polybrene, which facilitates adsorption of DNA to the cell surface. Expression of the transfected gene is induced by subjecting recipient cells to heat shock conditions. CAT activity in lysates from transfected cells is assayed using a simple thin layer chromatographic procedure. Adaptation of the transfection protocol to other insect cell types is discussed.     

Non-viral bone morphogenetic protein 2 transfection of rat dental pulp stem cells using calcium phosphate nanoparticles as carriers

Calcium phosphate nanoparticles have shown potential as non-viral vectors for gene delivery. The aim of this study was to induce bone morphogenetic protein (Bmp)2 transfection in rat dental pulp stem cells using calcium phosphate nanoparticles as a gene vector and then to evaluate the efficiency and bioactivity of the transfection. We also intended to investigate the behavior of transfected cells when seeded on 3-dimensional titanium fiber mesh scaffolds. Nanoparticles of calcium phosphate encapsulating plasmid deoxyribonucleic acid (DNA) (plasmid enhanced green fluorescent protein-BMP2) were prepared. Then, STRO-1-selected rat dental pulp stem cells were transfected using these nanoparticles. Transfection and bioactivity of the secreted BMP2 were examined. Thereafter, the transfected cells were cultured on a fibrous titanium mesh. The cultures were investigated using scanning electron microscipy and evaluated for cell proliferation, alkaline phosphatase activity and calcium content. Finally, real-time polymerase chain reaction was performed for odontogenesis-related gene expression. The results showed that the size of the DNA-loaded particles was approximately 100 nm in diameter. Nanoparticles could protect the DNA encapsulated inside from external DNase and release the loaded DNA in a low-acid environment (pH 3.0). In vitro, nanoparticle transfection was shown to be effective and to accelerate or promote the odontogenic differentiation of rat dental pulp stem cells when cultured in the 3-dimensional scaffolds. Based on our results, plasmid DNA-loaded calcium phosphate nanoparticles appear to be an effective non-viral vector for gene delivery and functioned well for odontogenic differentiation through Bmp2 transfection.     

Energy dependence of DNA-mediated gene transfer and expression

When cells are transfected with calcium phosphate-precipitated DNA, most of the DNA is internalized through active endocytosis but a small pool is postulated to be internalized by passive diffusion. The relative importance of these two DNA pools in gene transfer and expression has never been examined. We now show that mouse Ltk^– fibroblasts internalize >90% of radioactively labeled, calcium phosphate-precipitated DNA by a temperature- and energy-dependent process but a small pool of 2–3% of radioactively labeled DNA may be internalized even at 4° C or under energy depletion conditions. The contribution of these two pools of DNA to gene expression was examined by transfecting Ltk^– cells with DNA from a plasmid encoding marker for both transient expression (human growth hormone) and stable integration (neomycin resistance). When cells were depleted of ATP during transfection, transient expression of human growth hormone was reduced to only 2% of control values at day 4 posttransfection. Under the same condition, stable expression of neomycin resistance also was reduced to <1% of the control. Therefore, these data showed that only the pool of calcium phosphate-precipitated DNA internalized through an energy-dependent process is ultimately responsible for transient and stable expression of exogenous genes.     

重组人肝细胞生长因子在人脐带间质干细胞中高效表达

目的：构建能表达人肝细胞生长因子(hHGF) 的pWPI-hHGF载体，并将含hHGF基因的重组慢病毒载体转染人脐带间质干细胞（hUCMSCs），观察人脐带间质干细胞中hHGF的表达情况。方法：将携带目的基因hHGF的 pUC-SRα/hHGF质粒亚克隆到真核细胞表达载体pWPI载体上，构建重组质粒pWPI-hHGF。基因测序进行HGF的鉴定。用磷酸钙沉淀法，将pWPI-hHGF、pAX2、pMD2G共同转染包装细胞293T细胞，获得携带目的基因hHGF和GFP基因的重组慢病毒pWPI-hHGF。将pWPI-hHGF及阳性对照质粒pWPI-GFP分别用Lipofectamin 2000介导转染体外培养的hUCMSCs。在荧光显微镜下计数，确定阳性对照质粒的转染数，从而估计该基因的转染效率。蛋白印迹法检测HGF和GFP蛋白的表达，ELISA法检测细胞上清液hHGF含量。结果：DNA 测序显示hHGF基因成功地插入到pWPI载体中。包装细胞293T转导pWPI-HGF质粒后，转染阳性率达100％。阳性对照质粒转染人脐带间质干细胞24 h后，在荧光显微镜下观察，其转染效率达80%以上。蛋白印迹法检测靶细胞中hHGF表达呈强阳性，而对照组表达量极低，两者差异显著(P<0.01)。检测收集转染hHGF后的人间质干细胞上清液中hHGF的表达含量明显高于对照组(P<0.01)。结论：构建的在真核细胞内表达hHGF的重组质粒pWPI-hHGF转染人脐带间质干细胞，能获得hHGF基因在人脐带间质干细胞中大量、稳定的表达。     

Fast and Efficient Transfection of Mouse Embryonic Stem Cells Using Non-Viral Reagents

Reliable and efficient DNA and RNA transfection methods are required when studying the role of individual genes in mouse pluripotent stem cells. However, these cells usually grow in tight clusters and are therefore more difficult to transfect than many other cell lines. We have found that transfection is especially challenging when mouse embryonic stem (mES) cells are cultured in the newly described 2i medium, which is based on two chemical inhibitors of differentiation pathways. In the present study we have performed a side-by-side comparison of commercially available, non-viral transfection reagents with regard to their ability to deliver plasmid DNA and siRNA into adherent and/or trypsinized mES cells cultured in 2i medium, assessing transfection rates, plasmid gene expression, siRNA mediated knockdown of Oct4 and viability. Finally, we present a fast and efficient method for transfection of trypsinized mES cells using the liposomal-based Lipofectamine 2000. With only a five-minute long transfection time we obtained at least 85 % transfected cells with 80 % maintained viability. Moreover, this protocol saves up to a day of experimental time since the cells are in suspension at the time of transfection, which allows for immediately re-plating into the appropriate format. This fast, simplified and highly efficient transfection method will be valuable for both basic research and high-throughput applications.     

Transfection of cloned Heliothis zea cell lines with the DNA genome of the Heliothis zea nuclear polyhedrosis virus

Transfection conditions were optimized for the cloned UND-K derivative of the IPLB-HZ 1075 cell line using the calcium-phosphate co-precipitation technique and the DNA genome of the Heliothis zea S-type nuclear polyhedrosis virus. Optimal efficiencies were obtained using supercoiled viral DNA, and by extending the adsorption period for the diluted precipitate to 12 h. Transfection efficiencies ranging from 0.5 to 1.3 x 10(3) plaque forming units per microgram of supercoiled viral DNA were routinely obtained for UND-K cells and HzS-15 viral DNA. Transfection efficiencies were compared for 10 other cloned Heliothis cell strains and the uncloned parental IPLB-HZ 1075 cell line. The cloned cell strains UND-F, L, and U were incapable of transfection, while UND-I and G were 3 and 131 fold (respectively) less efficient than UND-K. The UND-K cells and the calcium phosphate transfection procedure permit relatively efficient in vitro manipulation of the Heliothis zea NPV virus genome.     

Simple, efficient, and reproducible gene transfection of mouse embryonic stem cells by magnetofection

Embryonic stem (ES) cells are recognized as an excellent cell culture model for studying developmental mechanisms and their therapeutic modulations. The aim of this work was to define whether using magnetofection was an efficient way to manipulate stem cells genetically without adversely affecting their proliferation or self-renewal capacity. We compared our magnetofection results to those of a conservative method using FuGENE 6. Using enhanced green fluorescent protein (eGFP) as a reporter gene in D3 mouse ES (mES) cells, we found that magnetofection gave a significantly higher efficiency (45%) of gene delivery in stem cells than did the FuGENE 6 method (15%), whereas both demonstrated efficient transfection in NIH-3T3 cells (60%). Although the transfected D3 (D3-eGFP) mES cells had undergone a large number of passages (>50), a high percentage of cells retained ES markers such as Oct-4 and stage-specific embryonic antigen-1 (SSEA-1). They also retained the ability to form embryoid bodies and differentiated in vitro into cells of the three germ layers. eGFP expression was sustained during stem cell proliferation and differentiation. This is the first transfection report using magnetofection in ES cells. On the basis of our results, we conclude that magnetofection is an efficient and reliable method for the introduction of foreign DNA into mouse ES cells and may become the method of choice.     

Phleomycin resistance as a dominant selectable marker in CHO cells

The Tn5 and the Streptoalloteichus hindustanus (Sh) ble genes conferring resistance to bleomycin-phleomycin antibiotics have been cloned into a mammalian vector under the RSV-LTR promoter. The resulting plasmids, pUT506 and pUT507 respectively, were used to transfect CHO cells by either the calcium phosphate or the recently described polybrene-DMSO method. Phleomycin- or bleomycin-resistant clones arose with a higher frequency after transfection with pUT507, and pUT507 transfectants were more resistant to both antibiotics than pUT506 transfectants. Phleomycin resistance in pUT507 transfectants was stable and associated with integration of plasmid sequences in genomic DNA. The Sh ble gene, which confers a dominant phleomycin-resistance phenotype, should provide a useful transferable selectable marker in CHO cells as well as in other animal cell lines.     

Restoration of cyclic adenosine monophosphate-stimulated chloride channel activity in human cystic fibrosis tracheobronchial submucosal gland cells by adenovirus-mediated and cationic lipid-mediated gene transfer.

In human airways, the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is predominantly expressed in serous cells of the tracheobronchial glands. Despite considerable evidence that submucosal glands are important contributors to the pathophysiology of CF lung disease, most attempts at CFTR gene transfer have primarily targeted airway surface epithelial cells. In this study, we systematically evaluated CFTR gene transfer into cultures of immortalized CF human tracheobronchial submucosal gland (6CFSMEO) cells using adenovirus and cationic lipid vectors. We found that the efficiency of adenovirus-mediated gene transfer was comparable in 6CFSMEO and CFT1 cells (a surface airway epithelial cell line isolated from a subject with CF). So was the ranking order of adenovirus vectors containing different enhancers/promoters (CMV > E1a approximately phosphoglycerokinase), as determined by both X-Gal staining and quantitative measurement of beta-galactosidase activity. Further, we provide the first demonstration that cationic lipids mediate efficient gene transfer into 6CFSMEO cells in vitro. The transfection efficiency at optimal conditions was higher in 6CFSMEO than in CFT1 cells. Finally, either infection with adenoviral vectors or transfection with cationic lipid:plasmid DNA complexes encoding CFTR significantly increased chloride (Cl-) permeability, as assessed using the 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ) fluorescence assay, indicating restoration of functional CFTR Cl- channel activity. These data show that although the mechanisms of transfection may be different between the two cell types, 6CFSMEO cells are as susceptible as CFT1 cells to transfection by adenoviral and cationic-lipid gene transfer vectors.     

Efficacy of cationic liposome-mediated gene transfer to mesangial cells in vitro and in vivo

Mesangial cells represent a major target for gene transfer approaches to the kidney. To establish a liposome-based system for transfection of mesangial cells we analyzed the efficacy and toxicity of different cationic liposomes and other nonviral transfection methods in primary cultures of rat and human mesangial cells using the Escherichia coli beta-galactosidase (lacZ) gene as a marker. In addition, an expression vector containing a human renin cDNA under the control of the cytomegalovirus immediate-early promoter/enhancer was generated, introduced into mesangial cells, and assayed in a system of transient gene expression. In vivo, gene transfer was studied after infusion of liposome/DNA complexes in the kidney of rats via the renal artery. Transfection efficiency ranged from 5.5% with DMRIE Liposomes in rat mesangial cells to 1.1% with LipofectAmine liposomes in human mesangial cells. Cytotoxicity following transfection was dependent on the transfection method. Transfection with the human renin expression vector led to the secretion of 11 pg/10(4) cells/48 h human renin in rat mesangial cells, 3,600 pg/10(4) cells/48 h in 293 cells, and 113 pg/10(4) cells/48 h human renin in opossum kidney cells. In vivo, infusion of liposomes was accompanied by nephrotoxicity and did not result in marker gene expression. Together the data demonstrate that cationic liposomes are useful tools for transferring genes into mesangial cells, including human mesangial cells. Cationic liposomes provide a functional system for the synthesis and secretion of human renin in mesangial cells and other mammalian kidney cells. The current limitation of the evaluated liposomes for an efficient in vivo gene transfer to mesangial cells is the toxicity upon intrarenal arterial administration.     

Easy stable transfection of a human cancer cell line by electrogene transfer with an Epstein–Barr virus-based plasmid vector

We report an easy and stable transfection technique using electrogene transfer with a nonviral Epstein–Barr (EB) virus-based vector. To achieve stable transfection of human breast cancer cells, we conducted electrogene transfer of an EB virus-based plasmid vector (reduced size of oriP) containing the enhanced green fluorescence protein (eGFP) gene. Because the EB virus-based vector exhibits high transfer efficiency and strong persistent transgene expression as a result of autonomous replication in human cells, and as Nucleofector electrogene transfer can achieve highly efficient gene transfection, this method is particularly suitable for generation of stably transfected cell lines.     

Transient expression of virus-specific promoters in murine resident peritoneal macrophages

Monocyte-macrophages (MO), being non-permissive for most viruses, play an important role in resistance to virus infection. In order to establish the mechanism of abortive infection of murine resident peritoneal MO (ResPMO) by herpes simplex virus type 1 (HSV-1), it is desirable to transfect these cells with viral promoters linked to an assayable gene, for example, the bacterial chloramphenicol acetyl transferase (CAT) gene. This will facilitate studies designed to measure levels of promoter activation or repression in these specialized cells. Transient expression of CAT in ResPMO was achieved with DEAE-dextran, but not using either calcium phosphate precipitate or lipofectin. CAT expression driven by various virus-specific promoters was less efficient in ResPMO compared with Vero cells and approximately 50% of input plasmid DNA remained in Vero cells at 48 h post transfection, but only 9% was detectable in ResPMO. However, approximately 6% of ResPMO and 9% of Vero cells contained CAT-specific DNA at 24 h post transfection. In addition, 2% of cells of either cell type contained CAT-specific polypeptide at 48 h. This is therefore the first report that the non-replicating murine ResPMO can be transfected in vitro and more importantly, that these cells express the transfected gene products.     

Transfection optimization for primary human CD8+ cells

Electroporation, a non-virus-mediated gene transfection method, has traditionally had poor outcomes with low gene transfection efficiency and poor cellular viability, particularly in primary human lymphocytes. Herein we have optimized the electroporation conditions for primary CD8+ cells resulting in a maximum rate of 81.3%, and a mean transfection efficiency of 59.6%. After removal of dead cells, the viability of transfected primary CD8+ cells was greater than 90%, similar to untransfected controls. Using this procedure, primary human CD8+ cells transfected with an interferon α8 plasmid produced fluids that inhibited HIV-1 replication by > 95%. This transfection protocol is useful for transfection of other primary blood cells, such as CD4+ T cells, and for studying the function of genes in primary human blood cells instead of cell lines. The transfection procedure also has potential application in gene therapy clinical trials to treat diseases utilizing transfected primary human cells.     

Differential replication of SV40 and polyoma DNAs in Chinese hamster ovary cells

We have investigated the ability of CHO cells to allow growth of papovaviruses by analyzing viral DNA replication after transfection using the calcium-phosphate co-precipitation technique. These analyses showed that when SV40-containing plasmids were introduced into CHO cells, viral DNA replicated to a level of approximately 1000 copies per T antigen-expressing cell, and neither late proteins nor virus progeny were produced. When polyoma (Py)-containing plasmids were transfected into CHO cells, a ten-fold higher level of Py DNA was present per T antigen-positive cell, and viral capsid proteins and progeny virus were detected, indicating that CHO cells are not equally restricted for all papovaviruses. Infection with intact virions was restricted in both cases. These results indicate that either SV40 or Py DNA introduced into CHO cells are able to express their early viral functions, and that different interactions of cellular proteins involved in the replication machinery with viral nucleic acids and proteins result in different levels of viral DNA synthesis and virus progeny production. We propose that, because of their favorable genetic characteristics, CHO cells should, therefore, provide a valuable experimental system for definition of the cellular contributions to papovavirus replication.     

A calcium phosphate-based gene delivery system

Although nonviral vectors have lower transfection efficiency than viral vectors, the excellent safety profile of nonviral vectors is appealing for gene therapy. An efficient, simple nonviral vector gene delivery system has been designed that includes plasmid DNA-calcium phosphate precipitates (pDNA-CaP) and porous collagen spheres (Cultispherestrade mark). The hypothesis for this study was the pDNA-CaP would achieve efficient plasmid DNA transfection and the porous collagen spheres would provide a suitable delivery carrier system for three-dimensional (3D) administration. To test the hypothesis, plasmid DNA including the LacZ reporter gene encoding beta-galactosidase was precipitated with CaP to form particles of compacted LacZ-CaP and delivered directly or by Cultispherestrade mark to cells in vitro. The transfection efficiency was determined by beta-galactosidase gene expression. Results indicated that pLacZ-CaP promoted 25-84% of transfection efficiency in a broad cell line spectrum and in flexible experimental conditions. Maximum transfection efficiency was achieved by having mostly nano-sized partles (50-200 nm in diameter) of pDNA-CaP precipitates. Seeding density of 0.7-4 x 10(4) cells/cm2 provided sufficient transfection efficiency, and storage of pDNA-CaP at 4 degrees C was most efficient to preserve transfection efficacy for up to 3 days. The pDNA-CaP worked well in the presence of serum and serum-free conditions and was less cytotoxic than the liposomes. Cultispherestrade mark carrying plasmid LacZ-CaP was an effective 3D system for gene delivery. The technique described here is a simple and safe procedure to deliver genes, and may have application to regenerate bone and other tissues.     

Bone cell transfection in tissue culture using hydroxyapatite microparticles

Coprecipitates of calcium phosphate and DNA have been used in vitro for several decades for cell transfection. We evaluated the efficiency of calcium phosphate ceramics associated to plasmid DNA in the transfection of bone cells in vitro when they are grown in tissue culture. Newborn rat calvariae and tibia epiphyses were grown on an agar surface for a period of 48 h to 30 days. The hydroxyapatite (HA)-particles were loaded with a plasmid bearing a galactosidase reporter gene by incubation of the plasmid solution in PBS with the particles. One milligram of HA-particles was then placed in contact with the bone explants for 8 and 30 days. Histological sections were then performed and the galactosidase activity was revealed using an X-gal solution. At eight days, very few cells expressing the galactosidase activity were detected. By 30 days, however, the explants appeared uniformly stained blue. The staining of sections showed that the osteoblasts, chondroblasts, perichondroblasts, and perisoteal cells all expressed the lacZ gene while the number of cells stained in the control was negligible. The time dependence of the transfection suggests that transfection using ceramics is linked to the degradation of the ceramic by the cells. Furthermore, the cells are stained remote from the particles suggesting that the particles induce a coprecipitate of DNA in the explant.