144 Use of the CRISPR/CAS 9 system to produce porcine adipose-derived stem cells expressing enhanced green fluorescent protein

The goal of our project is to produce porcine adipose-derived stem cells (ASCs) stably expressing enhanced green fluorescent protein (eGFP) by using the clustered regularly interspaced short palindromic repeats (CRIPSR) technique. Fluorescent stem cells can facilitate the tracing and visualisation of stem cell migration, fusion, and participation in tissue regeneration after stem cell injection therapy, and represent a useful tool for tissue engineering research. The production of stem cells containing eGFP from ASCs using the CRISPR gene editing technique is able to reduce the time and labour requirement necessary for harvesting fluorescent cells from transgenic pigs. To generate fluorescent, edited cells, we utilised the ROSA 26 locus of pigs for insertion of the eGFP gene by homology-directed repair of Cas9-cleaved DNA at the ROSA 26 locus. The critical steps of producing stem cells expressing eGFP are (1) cloning of guide oligos into a Cas9 cutting vector and producing a repair template vector to insert GFP; (2) transfecting porcine stem cells with CRISPR plasmids; (3) cell sorting with flow cytometry to isolate colonies expressing GFP. A Rosa 26 Cas9-gRNA cutting vector was produced by cloning a guide RNA sequence into the vector backbone of plasmid pX458-GFP, and the donor vector was produced by the combination of the eGFP gene flanked with ROSA 26 genomic DNA inserted into plasmid pUC57. To isolate cells edited to contain the eGFP gene inserted into the ROSA-26 locus, we transfected 250,000 cells with a 1:1 mass mixture of Cas9-gRNA and eGFP repair plasmid using Lipofectamine STEM reagent (Invitrogen) in three trials. GFP+ cells were isolated by fluorescence-activated cell sorting, plated in 96-well plates, and monitored for colony growth and GFP expression. These trials produced an average of ∼70 colonies from sorting, and ∼1% GFP+ colonies. As pX458 drives expression of GFP as a marker for transfection, we hypothesised that we would potentially isolate more GFP+ edited colonies if we utilised a Cas9-gRNA cutting vector expressing mCherry and sorted for cells expressing both mCherry and GFP. This would allow enrichment of edited cells expressing GFP early after transfection, without interference of cells expressing GFP from the Cas9-gRNA vector alone. Utilising this method, we again obtained an average of ∼70 colonies from sorting, and 3% GFP+ colonies. Results were subjected to Student’s t-test. The comparisons were colonies/cell sorted and GFP+ colonies/cell sorted. All data were expressed as quadratic means+mean SE. When we compared groups, no differences were found for colonies/cell sorted: P=0.53 (1.11 E-03±9.16E-04 and 5.39 E-04±3.77 E-04, respectively, for green-green or red-green) and for GFP+ colonies/cell sorted: P=0.44 (1.94 E-05±2.15E-05 and 4.59 E-05±2.46 E-05, respectively, for green-green or red-green). In conclusion, our attempts to isolate ASC edited to express GFP have been successful, and our initial results suggest that utilising a dual fluorescent label sorting strategy does not enhance the number of GFP+ ASC colonies isolated. Future studies will verify that our GFP+ ASC retain normal stem cell properties.