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Abstract
Acyl-CoA synthetase 4 (Acsl4) is involved in lipid synthesis and fatty acid degradation, and disruption of its function causes lipid metabolism disorder in various species. Herein, we report the generation of Acsl4 knockout (KO) mice using the CRISPR/Cas9 gene editing system to study its effects on lipid deposition. In this report, a large 12kb deletion in the Acsl4 gene was performed by coinjection of Cas9 mRNA and two guide RNAs (sgRNAs) into mouse fertilized oocytes. Six mutant mice carrying target mutations were examined by PCR analysis and direct sequencing. The gene modified mice remained healthy and displayed normal behavior. All the mutant F0 mice were mated with wild mice to produce the F1 generation, and only 1 F1 mutant mouse was obtained.
Highlights
Porcine intramuscular fat (IMF) content is considered one of the most important traits of pork quality, and has been positively correlated with meat tenderness, moisture content and taste [1]
Mutations in the pig Acyl-CoA synthetase long chain family 4 (Acsl4) gene have been found in pigs with high IMF content, its prompts us to generate Acsl4 gene knockout mouse to model this phenomenon
We designed two sgRNAs targeted on exon 5 and exon 10 of the mouse Acsl4 gene (Figure 1) The sgRNA target sequence did not cross-react with any other sites in the mouse genome and was followed by an NGG protospacer adjacent motif
Summary
Porcine intramuscular fat (IMF) content is considered one of the most important traits of pork quality, and has been positively correlated with meat tenderness, moisture content and taste [1]. Chen (2014) reported that pig Acsl gene seems to be a candidate gene to improve IMF content in pig breeding system, because of the association of the RsaI polymorphism and IMF in 2 native Chinese pig breeds(Yanan and Jinhua pigs) and DLY (Duroc x(Landrace x Yorkshine) pigs [4] All these studies suggest that Acsl is an ideal target for increasing IMF in livestock, but the detail mechanism is still not clear. Animal model with targeted gene deleted provide us important tool for gene function analysis, while it is very difficult for us to produce a geneknockout pig model because of the costs associated with housing and maintaining large animals and long generation interval, even if the CRISPR/Cas system has been widely used and had greatly simplified the program. It is the first Acsl knockout (KO) mouse model to allow the functional analysis of Acsl gene in mammalian vertebrate, and it can be applied for screening lipid metabolism related genes
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