, 2007 and Roy and Hart, 2010). An alternative and emerging method of gene targeting is mediated by zinc finger nucleases. Zinc finger nucleases are chimeric proteins generally consisting of
three zinc finger domains, each recognizing a nucleotide triplet, fused to a Fok1 nuclease domain. They function as a dimer. Zinc finger nucleases have been used to generate mutations by nonhomologous end joining (Bibikova et al., 2002 and Beumer et al., 2006) or homologous recombination with an ectopic template as a substrate (Bibikova et al., 2003 and Beumer et al., 2006). Creating mutations via zinc finger nucleases seems attractive, especially since an embryo injection protocol has been established (Beumer et al., 2008). However, the method is not widespread and not all loci can be targeted. Critical information about genes and the proteins they encode is their cellular and subcellular distribution. find more These data are typically determined by in situ hybridization experiments and immunohistochemical
stainings using antibodies raised selleck inhibitor against the protein encoded by the gene. However, several powerful genetic methods are now available to visualize protein expression patterns through tagging of genomic rescue constructs, gene targeting, and protein trapping. Generally, genomic rescue constructs are obtained by traditional cloning into plasmids that are compatible with P element transgenesis ( Rubin and Spradling, 1982, Spradling and Rubin, 1982 and Le et al., 2007) or ΦC31-mediated site-specific integration ( Groth et al., 2004 and Bischof et al., 2007). A valuable alternative to generate tagged genomic rescue constructs emerged recently through recombineering ( Sharan et al., 2009). Recombineering can be performed with different recombination templates such as PCR products that encompass protein tags or oligonucleotides that encompass specific mutations ( Sharan et al., 2009).
Recombineering was first introduced into the Drosophila field as a versatile transgenic platform named P[acman] (P/ΦC31 artificial chromosome for manipulation) ( Venken et al., 2006). Recombineering can be used to retrieve small to large DNA fragments containing genes from existing genome-wide BAC libraries for Drosophila melanogaster via gap-repair ALOX15 in Escherichia coli. The ΦC31 integrase integrates the attB containing constructs into defined attP containing docking sites ( Groth et al., 2004, Venken et al., 2006, Bischof et al., 2007 and Markstein et al., 2008). Subsequent recombineering steps can then be performed to introduce changes that include point mutations and deletions in Escherichia coli for structure/function analysis ( Pepple et al., 2008 and Leonardi et al., 2011). Alternatively, different tags for visualization of protein expression, subcellular protein localization, or acute protein inactivation using FIASH-FALi ( Venken et al., 2008 and Kasprowicz et al.