Oligo annealing and cloning into the PX330 plasmid to drive gRNA expression was performed as described previously [9 ] using Addgene plasmid 42230 [http://www.addgene.org/42230/ , and reference 20 (link)]. Oligo pairs for the targeted genes are: GGTA1 (NCBI Accession: XM_005660398.1) 5’- CACCGAGAAAATAATGAATGTCAA-3’ (forward), 5’-AAACTTGACATTCATTATTTTCTC-3’ (reverse); CMAH (NCBI Accession: NM_001113015.1) 5’-CACCGAGTAAGGTACGTGATCTGT-3’ (forward), 5’-AAACACAGATCACGTACCTTACTC-3’ (reverse); β4GalNT2 (NCBI Accession: NM_001244330.1) 5’-CACCGTGTATCGAGGAACACGCTT-3’ (forward), 5’-AAACAAGCGTGTTCCTCGATACAC-3’ (reverse).
Liver derived cells [21 (link)] were co-transfected with all three gRNA/Cas9 plasmids. After 48 hours, the treated cells were passed over an IB4 lectin column to isolate αGal null cells [6 (link)]. Two million α-Gal negative cells were further stained with fluorescein labeled Dolichos biflorus Agglutinin (DBA)-FITC (Vector Laboratories, Burlingame, CA, USA) at 2 ug/ml in 500ul HBSS with 0.5% BSA and flow sorted for DBA negative cells using a BD FACSAria sorter (BD Bioscience, San Jose, CA). The presence of Neu5Gc, an indicator of CMAH gene function, was not analyzed prior to somatic cell nuclear transfer (SCNT).
Liver derived cells [21 (link)] were co-transfected with all three gRNA/Cas9 plasmids. After 48 hours, the treated cells were passed over an IB4 lectin column to isolate αGal null cells [6 (link)]. Two million α-Gal negative cells were further stained with fluorescein labeled Dolichos biflorus Agglutinin (DBA)-FITC (Vector Laboratories, Burlingame, CA, USA) at 2 ug/ml in 500ul HBSS with 0.5% BSA and flow sorted for DBA negative cells using a BD FACSAria sorter (BD Bioscience, San Jose, CA). The presence of Neu5Gc, an indicator of CMAH gene function, was not analyzed prior to somatic cell nuclear transfer (SCNT).