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Transfection Rate (transfection + rate)

Distribution by Scientific Domains

Medical Sciences	50%

Selected Abstracts

Stable transgene expression in human embryonic stem cells after simple chemical transfection

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2009
Jun Liu
In this study we used plasmid-based vectors to investigate the transcriptional activities of three commonly used promoters in transient and stable transfection of MEL-1, a human embryonic stem (ES) cell line, using ExGen500, Fugene HD, and Lipofectamine. We demonstrated that cytomegalovirus (CMV), phosphoglycerate kinase (PGK) and human elongation factor-1, (EF1,) promoters all resulted in robust activity of a reporter gene in MEL-1 ES cell transient transfections regardless of the transfection reagent. Stable transfection outcomes varied, depending on the promoter and the transfection reagent used in the study. The phenomenon of transgene silencing was observed, most notably with the CMV vector, with which no positive stably transfected clones were obtained. Of the methods used in the study, Fugene HD resulted in the highest stable transfection rate, estimated by antibiotic selection, with plasmids containing genes under the control of the EF1, or PGK promoters. Stably transfected cells maintained typical hES cell morphology, with immunostaining exhibiting expression of the hES cell markers: Oct4, SSEA4, Tra-1-60, and Tra-1-81. Further, embryoid bodies formed by suspension culture retained reporter gene expression. Following injection into immunodeficient mice, the transfected cell lines showed robust formation of teratomas with cell types representative of the three germ layers. Mol. Reprod. Dev. 76: 580,586, 2009. © 2008 Wiley-Liss, Inc. [source]

Screening the Structural and Functional Properties of Bicyclo-DNA: bcox -DNA

CHEMBIOCHEM, Issue 14 2008
Samuel Luisier
Abstract The synthesis of two novel pyrimidine bicyclonucleosides (bcox -nucleosides) has been accomplished. These bicyclonucleosides each carry a lipophilic benzyloxime substituent on the carbocyclic ring and show improved conformational similarity to 2,-deoxyribonucleosides as shown by their X-ray structures. The thymine-containing bcox -nucleoside was converted into the corresponding phosphoramidite building block and incorporated into oligodeoxyribonucleotides by standard phosphoramidite chemistry. Tm data with complementary RNA and DNA were measured and compared to corresponding cases of natural and unfunctionalized bc-DNA. It was found that single incorporations of bcox residues destabilize duplexes by roughly 5,°C per modification. The destabilization was found to be due to the oxime substituent and not to the bicyclic scaffold itself. No significant alteration of the base-pairing selectivity as a function of the modification was observed. With RNA (but not with DNA) as a complement the relative thermal destabilization of bcox -oligothymidylates was gradually reduced and converted into a stabilizing interaction with increasing numbers of consecutive modifications. While no cellular uptake of bcox -oligonucleotides into HeLa cells occurred without transfecting agents, a significant increase in the transfection rate relative to unmodified DNA was observed in complexation with lipofectamine. [source]

Polyurethane Scaffolds Seeded With Genetically Engineered Skeletal Myoblasts: A Promising Tool to Regenerate Myocardial Function

ARTIFICIAL ORGANS, Issue 2 2010
Britta Blumenthal
Abstract In animal models, intramyocardial injection of primary skeletal myoblasts is supposed to promote tissue regeneration and to improve cardiac function after myocardial infarction. The usage of genetically engineered myoblasts overexpressing the paracrine factors involved in tissue repair is believed to enhance these effects. However, cell therapy via injection is always accompanied by a high death rate of the injected cells. Here, we describe the construction of a growth factor-producing myoblast-seeded scaffold to overcome this limitation. Skeletal myoblasts were isolated and expanded from newborn Lewis rats. Cells were seeded on polyurethane (PU) scaffolds (Artelon) and transfected with DNA of VEGF-A, HGF, SDF-1, or Akt1 using the lipid-based Metafectene Pro method. Overexpression was verified by ELISA, RT-PCR (VEGF-A, HGF, and SDF-1) and Western blot analysis (Akt1). The seeded scaffolds were transplanted onto damaged myocardium of Lewis rats 2 weeks after myocardial infarction. Six weeks later, their therapeutic potential in vivo was analyzed by measurement of infarction size and capillary density. Primary rat skeletal myoblasts seeded on PU scaffolds were efficiently transfected, achieving transfection rates of 20%. In vitro, we noted a significant increase in expression of VEGF-A, HGF, SDF-1, and Akt1 after transfection. In vivo, transplantation of growth factor-producing myoblast-seeded scaffolds resulted in enhanced angiogenesis (VEGF-A, HGF, and Akt1) or a reduced infarction zone (SDF-1 and Akt1) in the ischemically damaged myocardium. In summary, we constructed a growth factor-producing myoblast-seeded scaffold which combines the beneficial potential of stem cell transplantation with the promising effects of gene-therapeutic approaches. Because this matrix also allows us to circumvent previous cell application drawbacks, it may represent a promising tool for tissue regeneration and the re-establishment of cardiac function after myocardial infarction. [source]

Gene transfer to ovine corneal endothelium

CLINICAL & EXPERIMENTAL OPHTHALMOLOGY, Issue 5 2001
Sonja Klebe MBBS
ABSTRACT Purpose: Modification of a donor cornea by gene therapy has potential to modulate irreversible rejection, the major cause of corneal graft failure. The sheep is a useful model for the human in this respect, as ovine endothelial cells are amitotic. The aim of the study was to investigate the ability of various non-viral and viral agents to transfer a reporter gene to ovine corneal endothelium. Methods: The non-viral agents Transfectin-10, Transfectin-20, Transfectin-50, SuperFect, Effectene and CLONfectin were used to deliver the reporter gene, Escherichia coli lacZ, to ovine corneal endothelium in vitro. A Herpes simplex virus-1 and an adenoviral vector each encoding E. coli lacZ were similarly tested. Infected corneas were organ-cultured for up to 7 days in vitro to allow transfection efficiency, duration of gene expression and toxicity attributable to each vector to be compared. Results: Scattered single or clusters of endothelial cells expressing the reporter gene were observed after transfection with CLONfectin, Transfectin-10, Transfectin-20 and Transfectin-50. SuperFect and Effectene were virtually in-effective. At best, the absolute number of infected cells per endothelial monolayer after 3 or 7 days of organ culture was estimated as < 0.01%. The Herpes simplex virus-1 vector also failed to transduce ovine corneal endothelium efficiently. In contrast, transfection rates of up to 70% of endothelial cells were observed with the adenoviral vector. Conclusion: Non-viral vectors and Herpes simplex virus-1 are unlikely to be suitable for gene therapy of corneal endothelium, because the efficiency of transfection is low compared with the rates achieved with adenoviral vectors. [source]