Generation of TALEN-mediated FH knockout rat model

Abstract

Transcription activator-like effector nucleases (TALENs) are valuable tools for precise genome engineering of laboratory animals. Here we utilized this technique for efficient site-specific gene modification to create a fumarate hydratase (FH) gene knockout rat model, in which there was an 11 base-pair deletion in the first exon of the FH gene in 111 rats. 18 live-born targeted mutation offsprings were produced from 80 injected zygotes with 22.5% efficiency, indicating high TALEN knockout success in rat zygots. Only heterozygous deletion was observed in the offsprings. Sixteen pairs of heterozygous FH knockout (FH+/−) rats were arranged for mating experiments for six months without any homozygous KO rat identified. Sequencing from the pregnant rats embryo samples showed no homozygous FH KO, indicating that homozygous FH KO is embryonically lethal. Comparatively, the litter size was decreased in both male and female FH+/− KO rats. There was no behaviour difference between the FH+/− KO and the control rats except that the FH+/− KO male rats showed significantly higher body weight in the 16-week observation period. Clinical haematology and biochemical examinations showed hematopoietic and kidney dysfunction in the FH+/− KO rats. Small foci of anaplastic lesions of tubular epithelial cells around glomeruli were identified in the FH+/− kidney, and these anaplastic cells were comparatively positive for Ki67, p53 and Sox9, and such findings are most probably related to the kidney dysfunction reflected by the biochemical examinations of the rats. In conclusion, we have successfully established an FH+/− KO rat model, which will be useful for further functional FH studies.

Apancreatic pigs cloned using Pdx1-disrupted fibroblasts created via TALEN-mediated mutagenesis

“…Given that various mutations in the human PDX1 gene give rise to a spectrum of disease phenotypes, ranging from the complete absence of the pancreas to maturity onset diabetes of the young [ 4 , 9 ], pigs with PDX1 gene mutations would be a useful animal model to study the association of diabetes mellitus and human pancreatic agenesis diseases. Transcription activator-like effector nuclease (TALEN)-mediated genome modification has been successfully applied to generate animals with monoallelic or biallelic gene modifications, such as mice [ 10 ], pigs [ 11 ], and even monkeys [ 12 ]. Therefore, the objective of this study was to produce PDX1 −/− pigs using a combination of TALEN technology and somatic cell nuclear transfer (SCNT).…”

Anti-senescence role of heterozygous fumarate hydratase gene knockout in rat lung fibroblasts in vitro

“…The isolated fibroblasts with the current method were rather homogeneous and were confirmed spindle in morphology, positive for vimentin and negative for α-SMA, supportive of fibroblasts. PCR products flanking the FH +/– mutation were sequenced three times with identical mutations as reported earlier [9]. Furthermore, diminished FH gene and protein expression was observed in the FH +/– cells.…”

“…We established a FH +/– rat model that was reported previously [9]. The purpose of our current study was to explore the impact of heterozygous FH knockout (KO) on metabolic reprogramming, cell biology and ageing in isolated lung fibroblasts in this rat model, with lung fibroblasts from normal rats as controls.…”

Applications of Genome Editing Technology in Animal Disease Modeling and Gene Therapy

“…Yu's work utilized TALENs for efficient genome editing in rat zygotes and obtained FH knockout rat offsprings. They established a novel rat model for further functional FH studies [54].…”