June 1, 2009
Drosophila Targeted Mutagenesis Featured In Cold Spring Harbor Protocols
High-throughput whole-genome analysis is becoming a standard laboratory approach for investigating cellular processes. Next-generation sequencing is replacing microarrays as the technique of choice for genome-scale analysis, because it offers advantages in both sensitivity and scale. The June issue of Cold Spring Harbor Protocols (www.cshprotocols.org/TOCs/toc6_09.dtl) features "Native Chromatin Preparation and Illumina/Solexa Library Construction" from Keji Zhao and colleagues at the National Heart, Lung and Blood Institute (http://public.nhlbi.nih.gov/Staff/Home/UserInputForPerson.aspx?OID=895&LabId=lmi). The article describes sample preparation for sequencing of chromatin-immunoprecipitated DNA (ChIP-Seq) to analyze histone modification patterns using native chromatin and the Solexa/Illumina Genome Analyzer. Step-by-step instructions are given for purification of human CD4+ T cells from lymphocytes and chromatin fragmentation using micrococcal nuclease (MNase) digestion, followed by chromatin immunoprecipitation (ChIP) and construction of a library for sequencing. The article is freely available on the website for Cold Spring Harbor Protocols (http://cshprotocols.cshlp.org/cgi/content/full/2009/6/pdb.prot5237).
Mutational analysis has long been a valuable tool for deciphering gene function. However, systematic repeated targeting of a single locus is difficult and is not a routine approach in multicellular organisms. Yikang Rong and colleagues at the National Cancer Institute (http://ccr.cancer.gov/Staff/Staff.asp?profileid=6230) have developed the Site-specific Integrase mediated Repeated Targeting (SIRT) method to facilitate targeted mutagenesis in Drosophila melanogaster. SIRT targets a landing site for the phage phiC31 integrase and allows the generation of several genetic variants at a locus of interest without having to perform multiple experiments. SIRT requires the construction of a series of plasmid vectors with varying arrangements of DNA elements. By taking advantage of bacterial recombineering approaches, SIRT bypasses the shortcomings of traditional cloning techniques that rely on the availability of convenient restriction enzyme cut sites. This method, "SIRT Combines Homologous Recombination, Site-Specific Integration, and Bacterial Recombineering for Targeted Mutagenesis in Drosophila," is freely accessible on the website for Cold Spring Harbor Protocols (http://cshprotocols.cshlp.org/cgi/content/full/2009/6/pdb.prot5236).
On The Net:
Cold Spring Harbor Laboratory