Gene targeting in Drosophila by zinc-finger nucleases
Kelly J. Beumera et al reported an efficient way of mutagenesis for any target in Drosophila by direct embryo injection of mRNAs encoding specific zinc-finger nucleases (ZFNs). To explore the consequences resulted from alterations in DNA forms the basis of genetic analysis and it can done in more than one way. In forward genetics, mutations responsible for particular phenotypes are traced back to their genomic location. In reverse genetics, a genomic target is identified and mutations are directed to it and this a remarkable technique in elucidating function of gene ,especially when any desired sequence change can be introduced.Gene targeting is a process where in a DNA molecule introduced into a cell replaces the corresponding chromosomal segment by homologous recombination, and thus offers a precise way to manipulate the genome. This method is used either to repair or inactivate any desired gene of interest.
Gene targeting relies on introduction of a double-stranded break (DSB) into a genomic locus to enhance the efficiency of recombination with an exogenously introduced homologous DNA “repair template”. Zinc finger nucleases are slowly getting immensely popular tools in targeting mutagenesis and provides excellent alternatives to homing endonucleases used earlier for creating site specific double stranded breaks. ZFNs consist of a DNA-binding zinc finger domain (composed of either three or four fingers) covalently linked to the non-specific DNA cleavage domain of the bacterial FokI restriction endonuclease. Each zinc finger motif is typically considered to recognise and bind to a three-base pair sequence and as such, a protein including more zinc fingers targets a longer sequence and therefore has a greater specificity and affinity to the target site. Depending upon the required specifications of the end-product, the included zinc fingers may be selected via a parallel, sequential or bipartite technique or through an in vitro cell-based technique. The key to successful targeting is the ability to design ZFNs for arbitrarily chosen targets.
In the present study published in december issue of PNAS Carroll’s lab presented an embryo injection method that yields new, targeted mutations by both homologous recombination and Nonhomologous end joining, and this they demonstrated by designing new pairs of ZFNs for targets in the Drosophila genes for coilin, a defining component of nuclear Cajal bodies and PAS kinase, a serine/threonine kinase that plays a role in metabolic regulation in yeast and mammals.
More on this topic :
Zinc finger consortium
Dana Carroll lab
Zinc Finger Nucleases @ Wikipedia
Reference article :
Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases.
Beumer KJ, Trautman JK, Bozas A, Liu JL, Rutter J, Gall JG, Carroll D.
Proc Natl Acad Sci U S A. 2008 Dec 16;105(50):19821-6. Epub 2008 Dec 8.
PMID: 19064913
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