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Direct colony cloning of adherent mammalian cells using rings or dilution cloning has been used frequently for obtaining stable transfectants after gene delivery. As an alternative to these methods, successful isolation of the cells in a single colony is possible by placing a trypsin-immersed small paper disk onto the colony and subsequently picking up the paper with the assumption that it carries the trypsinized cells. However, the cloning success using this technique largely relies on the cell type used. In the present study, a novel, simple, and non-invasive technique for the isolation of cells from single colonies using a disposable pipette tip was developed. Using this technique, success was achieved in isolating the clonal populations of genome-edited porcine fibroblastic cells with 100% efficiency after co-transfection with the clustered, regularly interspaced, short palindromic repeats-CRISPR associated protein 9 (CRISPR/Cas9)-based genome editing components [for targeting the porcine GGTA1 that encodes α-1,3-galactosyltransferase (α-GalT)] and the piggyBac-based gene delivery components [to enable efficient chromosomal integration of the transgene carrying the cDNA of enhanced green fluorescent protein (EGFP)]. A toxin-based, drug-free selection system involving saporin (plant toxin)-conjugated BS-I-B4 lectin (IB4SAP) was employed in the present study. Since IB4SAP binds specifically to the cell-surface α-Gal epitope (synthesized by α-GalT), it is supposed that treatment with IB4SAP theoretically eliminates the untransfected or genome-edited porcine cells with a mono-allelic knockout (KO) phenotype, while all the surviving clones have a bi-allelic GGTA1 mutation. A total of 16 clones were isolated in the present study, all of which exhibited loss of the α-Gal epitope (a cell-surface carbohydrate synthesized by α-GalT), suggesting that all the clones had a bi-allelic KO phenotype. Moreover, 75% of these clones expressed EGFP uniformly, while the remainder had mosaic or no EGFP expression. These findings indicate the fidelity of the developed pipette tip-aided cell cloning approach for the efficient isolation of genome-edited porcine fibroblast clones.

Development of a Novel Pipette Tip-Aided Cell Cloning Method for The Effective Isolation of Genome-Edited Porcine Cell