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Abstract
Complement protein C3 is the pivotal component of the complement system. Previous studies have demonstrated that C3 has implications in various human diseases and exerts profound functions under certain conditions. However, the delineation of pathological and physiological roles of C3 has been hampered by the insufficiency of suitable animal models. In the present study, we applied the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system to target the C3 gene in porcine fetal fibroblasts. Our results indicated that CRISPR/Cas9 targeting efficiency was as high as 84.7%, and the biallelic mutation efficiency reached at 45.7%. The biallelic modified colonies were used as donor for somatic cell nuclear transfer (SCNT) technology to generate C3 targeted piglets. A total of 19 C3 knockout (KO) piglets were produced and their plasma C3 protein was undetectable by western blot analysis and ELISA. The hemolytic complement activity and complement-dependent cytotoxicity assay further confirmed that C3 was disrupted in these piglets. These C3 KO pigs could be utilized as a valuable large animal model for the elucidation of the roles of C3.
Highlights
To date, several inherited C3 deficiencies in humans[20], guinea pigs[21], dogs[22], rabbits[23], and C3 knockout animal models[24] have been reported and have provided insights into understanding the diversified biological responses mediated by C3
clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated gene targeting of C3 in pig primary fetal fibroblast (PFFs)
To establish C3 knockout (KO) cell lines, the Cas9sgRNA1 targeting vector was co-transfected with a TD-tomato plasmid containing a neomycin resistance (Neo) gene into an early passage of primary PFFs that were derived from a 35-day-old male Bama mini fetus
Summary
Several inherited C3 deficiencies in humans[20], guinea pigs[21], dogs[22], rabbits[23], and C3 knockout animal models[24] have been reported and have provided insights into understanding the diversified biological responses mediated by C3. We successfully generated C3 targeted piglets by using the CRISPR/Cas[9] system combined with the somatic cell nuclear transfer (SCNT) technology These C3 KO pigs may serve as valuable large-animal models for further delineation of various functional roles played by C3
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