ZFN Service Zinc finger nucleases (ZFNs) is the oldest, and most researched approach in gene editing field. It allows rapid genome editing in a variety of cell types and different model organisms, and it makes profound impacts on biological and medical research in recent years.
Background of ZFN Zinc finger nucleases (ZFNs) are enzymes which comprise two units in its framework, DNA binding domains and cleavage domains (usually nucleases). Zincfinger DNA-binding proteins (ZFP) containing DNA binding domains are engineered to recognize specific target DNA sequences. The cutting enzyme, known as Fok1, is widely used nuclease in the ZFNs technology, which can introduce double-strand breaks (DSBs) and subsequent frame-shift mutations into genes, which can lead to their knockout. Usually, two ZFNs are constructed with each containing a half of the cutting enzyme-Fok1 to make DSBs in the double -helix DNA. Once ZFNs bind to either side of the targeted DNA, the two halves of Fok1 combine or dimerize, to make a cut between the two sets of zinc-finger proteins. By taking advantage of the fact that endogenous DNA repair machinery including non-homologous end joining (NHEJ) pathway and homology-directed repair (HDR) pathway, ZFNs can be used to precisely alter the genomes of cells and organisms. Because of the ability to disable dominant mutations in heterozygous individuals, ZFNs have been explored in human clinical trials, including genetic disorders such as cystic fibrosis and other complex diseases, including cancer, inherited neurological disorders, HIV infection, etc. In the gene therapy process, ZFN offers an excellent way for targeted delivery of the therapeutic genes to a pre-selected chromosomal site to produce corrected functions.
Fig.1 Schematic of ZFN knockout. (Wilson, 2008)
With the development of ZFNs, this innovative technology has attracted massive attention and has been applied to a variety of model organisms. It is very important to choose an appropriate donor vector in the ZFNs technology.
Stable Cell Line Engineering
Stable cell lines can overcome the low transfection efficiency of transient expression, but the construction process is time-consuming and cockamamie.
Target Validation Taking into consideration the potential off-target effects, target validation and evaluation the efficiency of gene modification are essential and critical phases of the gene editing process. The common methods include RT-PCR, quantitative RT-PCR, Western blot and FACS, etc. Creative Biolabs can also offer transgenic mice with TALEN-mediated genome modifications for our clients. Our transgenic mice have been used within the scientific community around the world to study the gene functions and establish numerous animal models of human disease.
Reference 1. Wilson, J.H. Knockout punches with a fistful of zinc fingers. Proceedings of the National Academy of Sciences. 2008, 105(15): 5653-5654.
Zinc finger nucleases (ZFNs) are enzymes which comprise two units in its framework, DNA binding domains and cleavage domains (usually nuclea...