Abstract
The transcription activator-like effector nucleases (TALENs) strategy has been widely used to delete and mutate genes in vitro. This strategy has begun to be used for in vivo systemic gene manipulation, but not in an organ-specific manner. In this study, we developed a modified, highly efficient TALEN strategy using a dual-fluorescence reporter. We used this modified strategy and, within 5 weeks, we successfully generated kidney proximal tubule-specific gene Ttc36 homozygous knockout mice. Unilateral nephrectomy was performed on the 6-week-old founders (F0) to identify the knockout genotype prior to the birth of the offspring. This strategy was found to have little effect on reproduction in the knockout mice and inheritability of the knockout genotypes. The modified TALEN knockout strategy in combination with unilateral nephrectomy can be readily used for studies of gene function in kidney development and diseases.
Highlights
Genetic manipulations in genetically engineered mice have been widely used to study gene function and understand the roles of genes in human genetic diseases
Determination of the transcription activator-like effector nucleases (TALENs) activity using a dualfluorescence reporter in vitro To determine the activity of TALEN–Ttc36 before microinjecting the mouse embryo, we constructed a reporter plasmid based on two fluorescent proteins, ZsGreen and tdTomato (Figure 1B)
The TALEN targeted gene region was inserted into a partly duplicated and nonfunctional ZsGreen gene followed by a sequence of internal ribozyme entry sites (IRESs) and a tdTomato gene
Summary
Genetic manipulations in genetically engineered mice have been widely used to study gene function and understand the roles of genes in human genetic diseases. TALENs are precise and efficient genomic engineering tools, which have been successfully used in many fields of scientific research [1]. They are fusion proteins and work in pairs, consisting of a modular DNA-binding domain and a FokI endonuclease monomer [2,3]. The DNA break can either be repaired by non-homologous endjoining (NHEJ) or homologous recombination (HR), which results in deletion/insertion mutations and specific site mutations or specific sequence additions, respectively. The conventional TALENs screening systems are mainly based on b-galactosidase or a single fluorescence reporter [7,8], which functions in an indirect manner and limits the assessment of the transfection efficiency
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