Double knock-in pig models with elements of binary Tet-On and phiC31 integrase systems for controllable and switchable gene expression

Inducible expression systems are indispensable for precise regulation and in-depth analysis of biological process.Binary Tet-On system has been widely employed to regulate transgenic expression by doxycycline.Previous pig models with tetracycline regulatory elements were generated through random integration.This process often resulted in uncertain expression and unpredictable phenotypes,thus hindering their applications.Here,by precise knock-in of binary Tet-On 3G elements into Rosa26 and Hipp11 locus,respectively,a double knock-in reporter pig model was generated.We characterized excellent properties of this system for controllable transgenic expression both in vitro and in vivo.Two att P sites were arranged to flank the td Tomato to switch reporter gene.Single or multiple gene replacement was efficiently and faithfully achieved in fetal fibroblasts and nuclear transfer embryos.To display the flexible application of this system,we generated a pig strain with Dox-inducing h KRASexpression through phiC31 integrase-mediated cassette exchange.After eight months of Dox administration,squamous cell carcinoma developed in the nose,mouth,and scrotum,which indicated this pig strain could serve as an ideal large animal model to study tumorigenesis.Overall,the established pig models with controllable and switchable transgene expression system will provide a facilitating platform for transgenic and biomedical research.

Improving the Cpf1-mediated base editing system by combining dCas9/dead sgRNA with human APOBEC3A variants

Base editor-mediated C-to-T base conversion obviates the requirements of double-strand breaks,thereby showing promise as a tool for disease modeling and gene therapy(Gaudelli et al.,2017;Rees and Liu 2018).The most actively used base editor comprises a Cas9 nickase(nCas9)with cytidine deaminase and fused uracil DNA glycosylase inhibitor at the carboxy terminus of nCas9 to inhibit uracil N-glycosylase effects(Pearl,2000;Kunz et al.,2009;Rees and Liu 2018).