Liposome‐mediated transfection of mature taste cells

The introduction and expression of exogenous DNA in neurons is valuable for analyzing a range of cellular and molecular processes in the periphery, e.g., the roles of transduction‐related proteins, the impact of growth factors on development and differentiation, and the function of promoters specific to cell type. However, sensory receptor cells, particularly chemosensory cells, have been difficult to transfect. We have successfully introduced plasmids expressing green and Discosoma Red fluorescent proteins (GFP and DsRed) into rat taste buds in primary culture. Transfection efficiency increased when delaminated taste epithelium was redigested with fresh protease, suggesting that a protective barrier of extracellular matrix surrounding taste cells may normally be present. Because taste buds are heterogeneous aggregates of cells, we used α‐gustducin, neuronal cell adhesion molecule (NCAM), and neuronal ubiquitin carboxyl terminal hydrolase (PGP9.5), markers for defined subsets of mature taste cells, to demonstrate that liposome‐mediated transfection targets multiple taste cell types. After testing eight commercially available lipids, we identified one, Transfast, that is most effective on taste cells. We also demonstrate the effectiveness of two common “promiscuous” promoters and one promoter that taste cells use endogenously. These studies should permit ex vivo strategies for studying development and cellular function in taste cells. © 2005 Wiley Periodicals, Inc. J. Neurobiol, 2005     

Synthesis of aminated polysorbate 80 for polyplex‐mediated gene transfection

To develop novel gene delivery carriers, aminated polysorbate 80 (P80‐NH₂) was synthesized with strong positively charged properties through the introduction of three amine groups. The resulting P80‐NH₂ and DNA polyplex exhibited superb condensation abilities due to the high densities of positively charged amines groups. Size and surface charge of polyplex were shown to be well suited for cellular internalization. In addition, the P80‐NH₂/DNA polyplex demonstrated acceptable transfection efficiency in HeLa cells and was nontoxic relative to the conventional 25‐kDa polyethyleneimine system. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Microbubble‐mediated sonoporation for highly efficient transfection of recalcitrant human B‐ cell lines

Sonoporation has not been widely explored as a strategy for the transfection of heterologous genes into notoriously difficult‐to‐transfect mammalian cell lines such as B cells. This technology utilizes ultrasound to create transient pores in the cell membrane, thus allowing the uptake of extraneous DNA into eukaryotic and prokaryotic cells, which is further enhanced by cationic microbubbles. This study investigates the use of sonoporation to deliver a plasmid encoding green fluorescent protein (GFP) into three human B‐cell lines (Ramos, Raji, Daudi). A higher transfection efficiency (TE) of >42% was achieved using sonoporation compared with <3% TE using the conventional lipofectamine method for Ramos cells. Upon further antibiotic selection of the transfected population for two weeks, we successfully enriched a stable population of GFP‐positive Ramos cells (>70%). Using the same strategy, Raji and Daudi B cells were also successfully transfected and enriched to 67 and 99% GFP‐positive cells, respectively. Here, we present sonoporation as a feasible non‐viral strategy for stable and highly efficient heterologous transfection of recalcitrant B‐cell lines. This is the first demonstration of a non‐viral method yielding transfection efficiencies significantly higher (42%) than the best reported values of electroporation (30%) for Ramos B‐cell lines.     

RS‐1 enhances CRISPR‐mediated targeted knock‐in in bovine embryos

Targeted knock‐in (KI) can be achieved in embryos by clustered regularly interspaced short palindromic repeats (CRISPR)‐assisted homology directed repair (HDR). However, HDR efficiency is constrained by the competition of nonhomologous end joining. The objective of this study was to explore whether CRISPR‐assisted targeted KI rates can be improved in bovine embryos by exposure to the HDR enhancer RS‐1. In vitro produced zygotes were injected with CRISPR components (300 ng/µl Cas9 messenger RNA and 100 ng/µl single guide RNA against a noncoding region) and a single‐stranded DNA (ssDNA) repair template (100 ng/µl). ssDNA template contained a 6 bp XbaI site insert, allowing targeted KI detection by restriction analysis, flanked by 50 bp homology arms. Following microinjection, zygotes were exposed to 0, 3.75, or 7.5 µM RS‐1 for 24 hr. No differences were noted between groups in terms of development or genome edition rates. However, targeted KI rates were doubled in the group exposed to 7.5 µM RS‐1 compared to the others (52.8% vs. 25% and 23.1%, for 7.5, 0, and 3.75 µM, respectively). In conclusion, transient exposure to 7.5 µM RS‐1 enhances targeted KI rates resulting in approximately half of the embryos containing the intended mutation, hence allowing direct KI generation in embryos.     

The role of dextran conjugation in transfection mediated by dextran-grafted polyethylenimine

BACKGROUND: Conjugation through primary amines is one of the most commonly used methods to modify polycationic vectors for gene delivery. A better understanding of the effect of the conjugation on the mechanisms of transgene expression can help design efficient polycationic vectors. METHODS: Dextran with a molecular weight of 1500 was grafted onto polyethylenimine (PEI) to produce various degrees of grafting in an effort to investigate how the conjugation affected the mechanisms of transgene expression. Flow cytometry was employed to quantitate the cellular entry of plasmid and the level of transgene expression, which were measured using ethidium monoazide labeled plasmid and green fluorescent protein (GFP), respectively. The buffering capacity of the grafted PEI was determined by titration, and the integrity of the DNA-polymer complexes were examined by exposure to heparin. RESULTS: Grafting of dextran onto PEI was found to significantly diminish the cytotoxicity, buffering capacity, cellular entry, and the integrity of the DNA-polymer complexes. The reductions enlarged as the degree of grafting increased from 0 to 1.84%; however, at an optimal degree of grafting, the dextran-grafted PEI enhanced the percentages of GFP-positive cells to a level 3 times and 1.3 times of those mediated by unmodified PEI for CHO and MDA-MB-231 cells, respectively. CONCLUSIONS: These results demonstrated that the conjugation of dextran onto the primary amines of PEI inhibited the entry of plasmid across the cell membrane, but the change in the structures of the DNA-polymer complexes was able to promote transgene expression when the degrees of conjugation fell below 0.64%.     

Two different CC‐NBS‐LRR genes are required for Lr10‐mediated leaf rust resistance in tetraploid and hexaploid wheat

Comparative study of disease resistance genes in crop plants and their relatives provides insight on resistance gene function, evolution and diversity. Here, we studied the allelic diversity of the Lr10 leaf rust resistance gene, a CC‐NBS‐LRR coding gene originally isolated from hexaploid wheat, in 20 diploid and tetraploid wheat lines. Besides a gene in the tetraploid wheat variety ‘Altar’ that is identical to the hexaploid wheat Lr10, two additional, functional resistance alleles showing sequence diversity were identified by virus‐induced gene silencing in tetraploid wheat lines. In contrast to most described NBS‐LRR proteins, the N‐terminal CC domain of LR10 was found to be under strong diversifying selection. A second NBS‐LRR gene at the Lr10 locus, RGA2, was shown through silencing to be essential for Lr10 function. Interestingly, RGA2 showed much less sequence diversity than Lr10. These data demonstrate allelic diversity of functional genes at the Lr10 locus in tetraploid wheat, and these new genes can now be analyzed for agronomic relevance. Lr10‐based resistance is highly unusual both in its dependence on two, only distantly, related CC‐NBS‐LRR proteins, as well as in the pattern of diversifying selection in the N‐terminal domain. This indicates a new and complex molecular mechanism of pathogen detection and signal transduction.