Tuning of endosomal escape and gene expression by functional groups, molecular weight and transfection medium: a structure–activity relationship study

A library of cationic polyacrylamide homopolymers was synthesized and their gene delivery, endosomal release, and interaction with endosome-specific lipids were investigated. The guanidinium-containing polymers were most efficient.

Gene silencing in bovine zygotes: siRNA transfection versus microinjection

The aim of this study was to compare gene silencing in bovine zygotes when small interfering RNAs (siRNAs) were introduced into bovine zygotes by microinjection or lipid-based transfection. In Experiment 1, E-cadherin siRNA was injected at 100 or 375 µM and compared with PBS-injected and non-injected controls. Embryos were then cultured in vitro for 7 days and periodically assessed for development. For transfection, zona-free zygotes were incubated in transfection medium with siRNA for 1 h at 39°C and then cultured to Day 7. Injection of PBS or 375 µM E-cadherin siRNA resulted in a decrease in the number of embryos reaching the 8-cell stage (51.5% and 45.5%) or the blastocyst stage (39.0 and 32.5%) compared with non-injected controls (62.9 and 45.0%, respectively; P < 0.05). Messenger RNA abundance was suppressed by 36 and 46% when siRNA targeting E-cadherin was injected at 100 and 375 µM, respectively, compared with controls (P < 0.05). Transfection with 100 nM E-cadherin siRNA decreased development to the 8-cell stage (20.3 versus 53.0%) and blastocyst stage (7.2 versus 18.2%) compared with controls (P < 0.05). Messenger RNA relative abundance was not different between controls (non-transfected or transfected with GAPDH or scrambled siRNA). However, transfection of zygotes with 100 and 200 nM E-cadherin siRNA led to a 72 and 38% reduction, respectively, in E-cadherin mRNA relative abundance in Day 7 blastocysts compared with controls (P < 0.05).

Effect of Osmolarity and Presence of Serum on the Efficiency of Cell Transfection Using Immunoporation

Immunoporation is a novel method of cell transfection based upon the use of a new type of beads, Immunofect beads, that can be targeted to make holes in different types of cells depending on the type of bead used. It is known that the efficiency of transfection of cells by some techniques can be affected by the presence of serum and another important factor that appears to affect transfection efficiency and cell viability is the osmolarity of the transfection medium. This report presents studies on the effects of serum and varying osmolarity on the efficiency of transfection using immunoporation. The results clearly indicate that in hypertonic media the presence of serum decreases the efficiency of transfection. In the case of osmolarity, increasing the osmolarity of the immunoporation medium increases the efficiency of transfection but above about 650 mOsm this increasing efficiency is offset by the much lower viability of the cells.

Hepatitis B viral core proteins with an N-terminal extension can assemble into core-like particles but cannot be enveloped

The structure of hepatitis B virus (HBV) nucleocapsids has been revealed in great detail by cryoelectron microscopy. How nucleocapsids interact with surface antigens to form enveloped virions remains unknown. In this study, core mutants with N-terminal additions were created to address two questions: (1) can these mutant core proteins still form nucleocapsids and (2) if so, can the mutant nucleocapsids interact with surface antigens to form virion-like particles. One plasmid encoding an extra stretch of 23 aa, including six histidine residues, fused to the N terminus of the core protein (designated HisC183) was expressed in Escherichia coli and detected by Western blot. CsCl gradient and electron microscopy analyses indicated that HisC183 could self-assemble into nucleocapsids. When HisC183 or another similar N-terminal fusion core protein (designated FlagC183) was co-expressed with a core-negative plasmid in human hepatoma cells, both mutant core proteins self-assembled into nucleocapsids. These particles also retained kinase activity. Using an endogenous polymerase assay, a fill-in HBV DNA labelled with isotope was obtained from intracellular nucleocapsids formed by mutant cores. In contrast, no such signal was detected from the transfection medium, which was consistent with PCR and Southern blot analyses. Results indicate that core mutants with N-terminal extensions can form nucleocapsids, but are blocked during the envelopment process and cannot form secreted virions. The mutant nucleocapsids generated from this work should facilitate further study on how nucleocapsids interact with surface antigens.