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A modified TALEN-based strategy for rapidly and efficiently generating knockout mice for kidney development studies.

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.

SUBMITTER: Liu Y

PROVIDER: S-EPMC3885652 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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A modified TALEN-based strategy for rapidly and efficiently generating knockout mice for kidney development studies.

Liu Yunhong Y Lv Xiaoyan X Tan Ruizhi R Liu Tianming T Chen Tielin T Li Mi M Liu Yuhang Y Nie Fang F Wang Xiaoyan X Zhou Puhui P Chen Mianzhi M Zhou Qin Q

PloS one 20140108 1

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. Unila ...[more]

PMID: 24416307

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Project description:RationaleTargeted genetic engineering using programmable nucleases such as transcription activator-like effector nucleases (TALENs) is a valuable tool for precise, site-specific genetic modification in the human genome.ObjectiveThe emergence of novel technologies such as human induced pluripotent stem cells (iPSCs) and nuclease-mediated genome editing represent a unique opportunity for studying cardiovascular diseases in vitro.Methods and resultsBy incorporating extensive literature and database searches, we designed a collection of TALEN constructs to knockout 88 human genes that are associated with cardiomyopathies and congenital heart diseases. The TALEN pairs were designed to induce double-strand DNA break near the starting codon of each gene that either disrupted the start codon or introduced a frameshift mutation in the early coding region, ensuring faithful gene knockout. We observed that all the constructs were active and disrupted the target locus at high frequencies. To illustrate the utility of the TALEN-mediated knockout technique, 6 individual genes (TNNT2, LMNA/C, TBX5, MYH7, ANKRD1, and NKX2.5) were knocked out with high efficiency and specificity in human iPSCs. By selectively targeting a pathogenic mutation (TNNT2 p.R173W) in patient-specific iPSC-derived cardiac myocytes, we demonstrated that the knockout strategy ameliorates the dilated cardiomyopathy phenotype in vitro. In addition, we modeled the Holt-Oram syndrome in iPSC-cardiac myocytes in vitro and uncovered novel pathways regulated by TBX5 in human cardiac myocyte development.ConclusionsCollectively, our study illustrates the powerful combination of iPSCs and genome editing technologies for understanding the biological function of genes, and the pathological significance of genetic variants in human cardiovascular diseases. The methods, strategies, constructs, and iPSC lines developed in this study provide a validated, readily available resource for cardiovascular research.

Dataset Information

A modified TALEN-based strategy for rapidly and efficiently generating knockout mice for kidney development studies.

Publications

A modified TALEN-based strategy for rapidly and efficiently generating knockout mice for kidney development studies.

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