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SPARC: Development of Pigs with mCherry‐CRE Recombinase Fusion Proteins in Cholinergic Neurons
Author(s) -
Sponchiado Mariana,
Liao Yan-Shin,
Collins Emily,
Whitworth Kristin M.,
Wells Kevin G.,
Prather Randall S.,
Reznikov Leah R.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.05788
Subject(s) - mcherry , cre recombinase , biology , green fluorescent protein , fusion protein , transfection , microbiology and biotechnology , transgene , cholinergic , genetics , genetically modified mouse , neuroscience , gene , recombinant dna
Large animals are increasingly utilized in biomedical research and offer an advantage in that their anatomy and physiology closely parallel humans. Yet despite these advantages, rodent models dominate many areas of research, including neuroscience. This might be due in part to the greater number of technologies available to rodent researchers. Here we describe the development of pigs with mCherry and CRE‐recombinase proteins expressed in cholinergic neurons. We first cloned a 5,972 bp DNA fragment containing the predicted choline acetyltransferase (ChAT) promoter from porcine genomic DNA and placed into a pGlow TOPO™ TA expression system. We then tested for promoter activity by transfecting the expression system into porcine hippocampal slices and assessed for green fluorescent protein (GFP). We observed GFP at 60–72 hours post transfection. Confirmation of specificity was achieved by using antibody detection of ChAT. To optimize the expression system, we relied upon proven strategies used in rodents, and added an additional 535 bp of predicted promoter to the 5’ end. We then used this sequence to drive production of a mCherry‐P2A‐CRE recombinase fusion protein (ChAT‐mCherry‐P2A‐CRE). We transfected brain slice cultures from the pig hippocampus, striatum and brainstem with ChAT‐mCherry‐P2A‐CRE. Approximately 60 hours later, we isolated RNA and froze brain slices from transfected and non‐transfected samples. qRT‐PCR revealed mCherry and CRE recombinase mRNA in all transfected tissues. Expression of mCherry protein was also visible in discreet subsets of neurons in transfected samples. Studies are ongoing to confirm mCherry expression in cholinergic neurons. Key next steps include engineering our ChAT‐mCherry‐P2A‐CRE to contain GGTA1 homology arms for placement into the GGTA1 safe harbor in the pig genome, providing in vivo validation of ChAT‐mCherry‐P2A‐CRE using lentivirus pseudotyped with rabies glycoprotein, and targeting porcine fetal fibroblasts for somatic cell nuclear transfer. If successful, this model will allow for simultaneous visualization of neurons (and their innervation), and the ability to control or monitor neural activity through CRE‐mediated recombination. Support or Funding Information HL119560, 1OT2OD026582