Reduction In Transfection Research Articles

Polymeric Gene Transfection on Insulin-Secreting Cells: Sulfonylurea Receptor-Mediation and Transfection Medium Effect

In vitro transfection of secreting cells is regarded as one strategy for improved cell engineering/ transplantation. Insulin-secreting insulinoma cell lines or pancreatic beta-cells could be genetically engineered using designed polymeric vectors which are safer than viral vectors. This study investigates the effects of the constituents in transfection media on polymeric transfection. Polyplexes conjugated with sulfonylurea (SU) were evaluated under different transfection conditions for gene transfection and their effects on cytotoxicity and insulin secretion. Several components in transfection media specifically associated with the insulin secretion pathway were amino acids, vitamins, Ca2+ and K+. The interactions of the polyplexes with insulin were monitored by surface charge and particle size to monitor how insulin as a protein influences transfection. For an insulin-secreting cell line (RINm5F), polyplexes in Ca2+--containing KRH medium (Ca2+(+)KRH) enhanced transfection and did not cause damage to biological functions. When adding amino acids, vitamins, or K+ or depleting Ca2+ from Ca2+(+)KRH, poly(L-lysine)/DNA complexes showed a greater reduction in transfection than SU receptor (SUR)-targeting polyplexes (SU-polyplex). Positively charged polyplexes interacted with insulin, developing a negative surface charge, and these interactions may cause a decrease in transfection. The findings suggest that in vitro and ex vivo polymeric transfection of insulin-secreting cells can be modulated and enhanced by adjusting the transfection conditions.

Positively charged synthetic peptides from structural proteins of papillomaviruses abrogate human papillomavirus infectivity.

Human papillomavirus (HPV) virus-like particles (VLP) and synthetic peptides corresponding to positively-charged sequences of the major and minor capsid proteins were tested for their efficacy in inhibiting the infectivity of HPV 31 pseudovirions by blocking virus entry into cells. A greater than 80% reduction of transfection was observed with one HPV-31 peptide at a concentration of 10 microg/ml. Moreover, the blocking was not type-specific since similar reduction in transfection was observed with peptides from other HPV types at a concentration of 60 microg/ml. This concentration was non-toxic for the cells. These findings indicate that some of the positively-charged sequences of the L1 and L2 HPV capsid proteins of papillomavirus are compounds that might be locally active against sexually transmitted papillomavirus. The findings provide further evidence that cellular glycosamino-glycans (GAGs) are functional receptors for HPVs.

Failure to detect a DNA repair-related defect in the transfection of ataxia-telangiectasia cells by enzymatically restricted plasmid.

We have transfected two SV40-transformed human fibroblast cell lines with plasmids in which double-strand breaks have been introduced by restriction enzymes, within or near the selected gene. Restriction of pSV2gpt with KpnI, as previously shown by Cox et al. (1986), reduced the frequency of transfection more in the ionizing radiation-sensitive ataxia-telangiectasia line AT5BIVA than in the resistant line MRC5V1. When the related plasmid pSV2neo was restricted with SmaI, the reduction in transfection was less in the ataxia-telangiectasia than in the normal cell line. Under our conditions, the apparent defect in transfection of AT5BIVA by pSV2gpt appears to be a result of the unusual sensitivity of the repair-deficient recipient to the selective agent. Loss of potential transfectants is exacerbated when transient gene expression is reduced by restriction of the plasmid. We suggest that a reduction in yield of transfectants with restricted plasmid in ataxia-telangiectasia cells cannot readily be used as evidence of a defect in DNA repair. Our results are also relevant to standard transfection experiments, since they emphasize the importance of optimizing selection when transient expression may be reduced, to ensure that potential transfectants are not killed by the selection regime.