Basic Cell Culture by Jeffrey W. Pollard, John M. Walker

By Jeffrey W. Pollard, John M. Walker

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2001), although the Ahringer RNAi feeding library makes RNAi by feeding the method of choice for high-throughput screening. Transgenic Expression of Plasmid(s) Producing Hairpin dsRNA RNAi is not effective in neurons. , 2000). , 2003). Tavernarakis et al. (2000) have described an RNAi method relying on dsRNA produced from a plasmid containing a heat-shock-inducible or tissue-specific promoter driving expression of an IR of a target gene (Fig. 2). Exon-rich genomic DNA (or cDNA) is amplified by using two primers that introduce unique restriction sites at the fragment ends.

There are four methods for double-stranded RNA (dsRNA) delivery in C. , 2000). In this chapter, we discuss the molecular genetic mechanisms, techniques, and applications of RNAi in C. elegans. Introduction C. elegans is a free-living, self-fertilizing hermaphroditic roundworm (Brenner, 1974). 5 mm), simple anatomy (<1000 cells), and ease of laboratory culturing make ‘‘the worm’’ a favorite genetic model organism (Barr, 2003). The constantly expanding molecular toolkit includes transgenesis, green fluorescent protein (GFP) to study gene expression and protein localization, primary cell culture, microarrays, and RNAi.

Silencing of FLC by using pHELLSGATE12. Rosette leaf number at flowering is shown for T1 plants of Arabidopsis ecotype C24 transformed with HG8-FLC, HG12-FLC-R (PDK intron reversed with respect to promoter), and HG12-FLC-F (PDK in forward orientation with respect to promoter); C24 wild type and flc20 (an flc null mutant) are also shown. Mean and standard deviation of rosette leaf numbers at flowering are also shown. TABLE I Phenotypes of T1 PDS-Silenced Plants Construct No. of transformants No phenotype Bleached cotyledons Some leaf bleaching Total bleaching HG8-PDS HG12-PDS-F HG12-PDS-R 8 15 5 1 (13%) 0 0 5 (63%) 6 (40%) 3 (60%) 2 (25%) 7 (47%) 2 (40%) 0 2 (13%) 0 PCR Amplification with attB Primers Primer sequences can be designed by using normal design rules with the addition of attB1 and attB2 sites to the 50 end of the forward and reverse primers, respectively (Fig.

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