Development of tools for genome engineering in swine.

Abstract

Heightened interest in relevant models for human disease, in the production of transgenic livestock for biomedical applications, and new and pending releases of genome sequences for rat, cow, and pig have increased the need for improved methods for germline transgenesis. Transpositional transgenesis (TnT) offers an efficient and precise mechanism for genome integration of transgene DNA that avoids incorporation of CG-rich vector DNA and multi-copy transgene concatemerization that can lead to suppression of gene expression and transgene instability. We have developed and tested a transposon toolbox (including Sleeping Beauty, Tol2, piggyBac, and Passport) in pig cells. We have also demonstrated the activity of Cre and Flp recombinases in cultured pig cells and have implemented that technology for regulated activation of gene expression in swine. The application of transposon and recombinase technologies significantly enhances both the means and possible complexity of pig genetic modification. The use of enhanced cis and trans components of the Sleeping Beauty (SB) transposon system for animal transgenesis by pronuclear injection (PNI) resulted in tremendous improvement in the creation of transgenic laboratory mice, rats, and pig embryos, increasing both the frequency of transgenic founders, and the number of transgenes per founder, overall elevating the number of potential transgenic lines by 10-20-fold. Genetic modification of pigs by tandem pig transgenesis and cloning also benefits from TnT, resulting in multiple independent insertions per cellular clone which permits the assessment of several alleles upon segregation from a single founder. Finally, towards the development of a porcine model of cystic fibrosis (CF), we’ve created a mouse phenocopy of CF based on RNA interference using the SB transposon system. Building on insights gained in CFTRRNAi mouse, we’ve developed tightly regulated CFTRRNAi alleles in pigs to circumvent the lethal neonatal meconium ileus that confounds the CFTR-knockout model, hopefully simplifying investigation of postnatal CFTR deficiency in a large animal. Given its simplicity, versatility and high efficiency, TnT represents a compelling non-viral approach to modifying the porcine germline.