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Transfection Methods (transfection + methods)
Selected AbstractsNucleofection: a new, highly efficient transfection method for primary human keratinocytes,EXPERIMENTAL DERMATOLOGY, Issue 4 2005Jörg H. W. Distler Abstract:, Transfection is an essential tool for numerous in vitro applications including studies of gene expression, promoter analysis, and intracellular signaling pathways and also for therapeutic strategies such as tissue engineering and gene therapy. However, transfection of primary cells including keratinocytes with common methods such as calcium phosphate, DEAE-dextran, liposome-mediated transfer, electroporation or viral vectors is problematic because of low transfection efficiency and the induction of terminal differentiation. Here we analyzed the use of nucleofection, a new, electroporation-based transfection method that enables the DNA to enter directly the nucleus, for the transfection of keratinocytes. Several different conditions were tested and optimized, resulting in a final transfection efficiency of 56% in primary human epidermal keratinocytes. This efficiency is superior to all non-viral transfection methods reported so far. The number of non-viable keratinocytes after nucleofection was low, varying between 14 and 16%. In contrast to other transfection protocols, nucleofection did not induce terminal differentiation in the transfected keratinocytes. In addition, nucleofection is a fast method, because the results can be analyzed within 7 h. In summary, nucleofection is a fast, easy and highly effective alternative for the transfection of primary human keratinocytes, which offers new opportunities for various research applications. [source] Uptake and Release of Double-Walled Carbon Nanotubes by Mammalian CellsADVANCED FUNCTIONAL MATERIALS, Issue 19 2010Vera Neves Abstract Efforts to develop carbon nanotubes (CNTs) as nano-vehicles for precise and controlled drug and gene delivery, as well as markers for in vivo biomedical imaging, are currently hampered by uncertainties with regard to their cellular uptake, their fate in the body, and their safety. All of these processes are likely to be affected by the purity of CNT preparation, as well as the size and concentration of CNTs used, parameters that are often poorly controlled in biological experiments. It is demonstrated herein that under the experimental conditions of standard transfection methods, DWNTs are taken up by cultured cells but are then released after 24 h with no discernable stress response. The results support the potential therapeutic use of CNTs in many biomedical settings, such as cancer therapy. [source] Genetically Manipulated Human Skeletal Myoblast Cells for Cardiac TransplantationJOURNAL OF CARDIAC SURGERY, Issue 6 2002Kh H Haider Aim: Considering the promise of skeletal myoblast cell transplantation to improve cardiac function in myocardial myopathies, we aim in the present study to investigate the potential of human skeletal myoblast cells (HSMC) as a carrier for therapeutic genes for the heart muscle. Methods: Skeletal muscle sample is obtained from rectus femoris of the donor and is processed in the tissue culture to generate HSMC by a patented process of Cell Therapy Inc. The HSMC are grown in large 225 mm2 tissue culture flasks coated with collagen for enhanced cell adherence, using patented Super Medium (Cell Therapy Inc., Singapore) containing 10% fetal calf serum, to 80% confluence. The HSMC are passaged at regular time intervals of 48-72 hours to prevent in vitro differentiation. The HSMC thus obtained are transduced three times with retroviral vector carrying Lac-Z reporter gene before transplantation. The Lac-Z transduced HSMC are harvested by trypsinization, washed and re-suspended in serum free Super Medium. Ischemic Porcine model is created by clamping ameroid ring around left circumflex coronary artery in Yorkshire swine, four weeks prior to cell transplantation. For cell transplantation, the animal is anaesthetized, ventilated and heart is exposed by left thoracotomy. Fifteen injections (0.25 ml each) containing 300 million cells are injected in to the left ventricle endocardially under direct vision. For control animal, only culture medium without cells is injected. The animal is euthanized at pre-determined time, heart is explanted and processed for histological examination. The cryosectioning of the tissue and subsequent staining for Lac-Z expression and Hematoxylin-Eosin staining is carried out by standard methods. Results: The skeletal muscle samples processed by the patented method of Cell Therapy yield 85-90% pure HSMC. The preliminary data shows that repeated transductions of myoblast cells with retrovirus carrying Lac-Z yield highly efficient 70-75% Lac-Z positive HSMC population (Figure 1). Dye exclusion test using Trypan blue reveals >95% cell viability at the time of injection. Gross sections of the cardiac tissue stained positive for Lac-Z expression (Figure 2). Histological examination showed the presence of grafted myoblast cells expressing Lac-Z gene in the cardiac tissue (Figure 3). Conclusion: In the light of our preliminary results, we conclude that HSMC may prove to be excellent carriers of transgene for cardiac muscle cells which otherwise are refractory to ordinary gene transfection methods. The use of HSMC mediated gene delivery to cardiac muscle is safer as compared to direct injection of viral vectors in to the heart muscle. Furthermore, the grafted myoblast cells will additionally serve to strengthen the weakened heart muscle. Figure 1.Human Skeletal myoblasts transduced with Lac-Z carrying retrovirus and stained with x-gal. Figure 2.Gross sections of heart muscle stained for Lac-Z expression. Figure 3.X-gal stained porcine heart muscle counter-stained with Eosin. The heart was explanted 6 weeks after transplantation of Lac-Z stained human myoblasts. The arrow shows Lac-Z expressing myoblast cells. [source] Auditioning of CHO host cell lines using the artificial chromosome expression (ACE) technologyBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2009Malcolm L. Kennard Abstract In order to maximize recombinant protein expression in mammalian cells many factors need to be considered such as transfection method, vector construction, screening techniques and culture conditions. In addition, the host cell line can have a profound effect on the protein expression. However, auditioning or directly comparing host cell lines for optimal protein expression may be difficult since most transfection methods are based on random integration of the gene of interest into the host cell genome. Thus it is not possible to determine whether differences in expression between various host cell lines are due to the phenotype of the host cell itself or genetic factors such as gene copy number or gene location. To improve cell line generation, the ACE System was developed based on pre-engineered artificial chromosomes with multiple recombination acceptor sites. This system allows for targeted transfection and has been effectively used to rapidly generate stable CHO cell lines expressing high levels of monoclonal antibody. A key feature of the ACE System is the ability to isolate and purify ACEs containing the gene(s) of interest and transfect the same ACEs into different host cell lines. This feature allows the direct auditioning of host cells since the host cells have been transfected with ACEs that contain the same number of gene copies in the same genetic environment. To investigate this audition feature, three CHO host cell lines (CHOK1SV, CHO-S and DG44) were transfected with the same ACE containing gene copies of a human monoclonal IgG1 antibody. Clonal cell lines were generated allowing a direct comparison of antibody expression and stability between the CHO host cells. Results showed that the CHOK1SV host cell line expressed antibody at levels of more than two to five times that for DG44 and CHO-S host cell lines, respectively. To confirm that the ACE itself was not responsible for the low antibody expression seen in the CHO-S based clones, the ACE was isolated and purified from these cells and transfected back into fresh CHOK1SV cells. The resulting expression of the antibody from the ACE newly transfected into CHOK1SV increased fivefold compared to its expression in CHO-S and confirmed that the differences in expression between the different CHO host cells was due to the cell phenotype rather than differences in gene copy number and/or location. These results demonstrate the utility of the ACE System in providing a rapid and direct technique for auditioning host cell lines for optimal recombinant protein expression. Biotechnol. Bioeng. 2009; 104: 526,539 © 2009 Wiley Periodicals, Inc. [source] Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfectionBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2008Sebastien Chenuet Abstract Transfection with polyethylenimine (PEI) was evaluated as a method for the generation of recombinant Chinese hamster ovary (CHO DG44) cell lines by direct comparison with calcium phosphate-DNA coprecipitation (CaPO4) using both green fluorescent protein (GFP) and a monoclonal antibody as reporter proteins. Following transfection with a GFP expression vector, the proportion of GFP-positive cells as determined by flow cytometry was fourfold higher for the PEI transfection as compared to the CaPO4 transfection. However, the mean level of transient GFP expression for the cells with the highest level of fluorescence was twofold greater for the CaPO4 transfection. Fluorescence in situ hybridization on metaphase chromosomes from pools of cells grown under selective pressure demonstrated that plasmid integration always occurred at a single site regardless of the transfection method. Importantly, the copy number of integrated plasmids was measurably higher in cells transfected with CaPO4. The efficiency of recombinant cell line recovery under selective pressure was fivefold higher following PEI transfection, but the average specific productivity of a recombinant antibody was about twofold higher for the CaPO4-derived cell lines. Nevertheless, no difference between the two transfection methods was observed in terms of the stability of protein production. These results demonstrated the feasibility of generating recombinant CHO-derived cell lines by PEI transfection. However, this method appeared inferior to CaPO4 transfection with regard to the specific productivity of the recovered cell lines. Biotechnol. Bioeng. © 2008 Wiley Periodicals, Inc. 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