SPARC: Development of a Transgenic Pig Expressing mCherry and CRE Recombinase Fusion Proteins Under the Control of the Murine Choline Acetyltransferase Promoter

Abstract

Pigs can serve as useful models for advancing the biomedical field due to anatomy and physiology that more closely resembles human anatomy and physiology compared to traditional rodent models. However, there is a paucity of porcine CRE-driver lines available, limiting their utility for refined neural mapping and neural modulation studies. Here we describe the development of pigs with mCherry and CRE recombinase proteins expressed in cholinergic neurons. A 6,419 bp DNA fragment containing the Mus musculus choline acetyltransferase (ChAT) promoter was used to drive production of a mCherry–P2A–CRE recombinase fusion protein (mmChAT–mCherry–P2A–CRE). Linearized mmChAT–mCherry–P2A–CRE plasmid DNA was introduced into male porcine fetal fibroblasts by electroporation. Positive colonies were selected with 400µg/mL G-418 and screened for transgene insertion by PCR. Two colonies showing targeted insertion were used for somatic cell nuclear transfer and the resulting blastocysts were transferred to surrogate gilts. Two early pregnancies were established, and one of them went to term. Six Duroc-Landrace male piglets were born by natural delivery. PCR analysis of genomic DNA showed that one piglet carried the transgene whereas the others were determined to be wild-type. The transgenic piglet was phenotypically normal with no obvious difference in appearance compared to the wild-type littermates. The piglet was euthanized at 7-weeks of age for further characterization. Major tissues known to have cholinergic cell bodies and cholinergic innervation (hippocampus, cerebellum, spinal cord, basal ganglion, dorsal root ganglion, nodose ganglion, vagus nerve, trachea, bronchus, heart, bladder, duodenum and pancreas) were evaluated regarding mCherry and CRE recombinase mRNA presence. End-point PCR revealed CRE recombinase mRNA in basal ganglion, nodose ganglion, dorsal root ganglion and heart. mCherry mRNA was found in bronchus homogenates. Studies are ongoing to confirm integration sites into the genome, and mCherry and CRE recombinase expression at the protein level in cholinergic neurons. If successful, this model will allow for simultaneous dissection of cholinergic circuit connectivity and control of neural activity through CRE-mediated recombination.