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A Novel Rat Model of Nonalcoholic Fatty Liver Disease Constructed Through CRISPR/Cas-Based Hydrodynamic Injection.
Molecular Biotechnology 2017 October
CRISPR/Cas technology has been widely used in generating conventional and conditional gene knockout animals through germline mutation. A recent study has demonstrated that CRISPR/Cas system also worked on nongermline mutation in mice liver via hydrodynamic injection of vector plasmid to blood circulation. However, whether this is also applied to rat and what is the optimal concentration of vector plasmid still need to be studied. Here, we attempted to use pX330-Pten plasmid to knock down the expression of Pten in rat liver for generation of nonalcoholic fatty liver disease (NAFLD) rat model. A range of pX330-Pten dosages (75, 150 and 300 μg/100 g) in 10 ml of saline water was injected into the blood circulation of SD rats within 30 s by tail vein injection. The data showed that, 9 weeks after injection, no differences of TC, AST and ALT were found between control and the treatment groups (low-dose, mid-dose, high-dose and fat-diet groups). However, the high-dose group revealed severe lipid deposition and significant knockdown of Pten, which was validated by western blot and real-time PCR. Notably, lipid deposition and Pten knockdown were detected in the liver of animals from the high-dose group as early as 6 weeks after injection. Taken together, our findings demonstrated that hydrodynamic injection of pX330-Pten plasmid at the dosage of 300 μg/100 g can knock down the expression of Pten in rat liver efficiently, resulting in lipid deposition as well as NAFLD. Taken together, this study presents a novel and efficient method to edit genome in rat liver.
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