P125

Highly efficient protocol to select null alpha 1,3-galactosyltransferase porcine cells

Luz M. Reyes, Jose L. Estrada, Bess Ivary, Leela L. Paris, Richard A. Sidner, A. Joseph Tector

IUPUI, Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, United States

Background: Cell selection to generate alpha 1,3-Galactosyltransferase (Gal) double knockout (DKO) pigs has been complex, inefficient and time-consuming. The objective of this study was to generate a highly efficient system to isolate Gal-DKO porcine liver derived cells (PLDC) using a combination of two sorting methods.

Materials and Methods: Gal single knockout (Gal-SKO) PLDC cells were cultured for seven to eleven passages. Thirty million Gal-SKO PLDC cells were stained with IB4-Alexafluor 488 and sorted for negative fluorescence. After sorting, cells were recovered in stem cell media (SCM) and cultured until confluent. A second selection was performed on the FACS-selected cells using biotin-conjugated IB4-lectin attached to streptavidin-coated magnetic beads. Gal-negative cells were collected in the supernatant and plated until colonies were formed.

Results: Forty-eight colonies developed; 44 (91.7%) were null colonies for the a-Gal epitope. Gal-DKO genotype was identified by PCR of genomic DNA of growing cells.

Conclusions: From cultured Gal-SKO cell lines previously developed in our lab, we isolated spontaneously mutated Gal-DKO cells. A combination of selection methods using flow cytometry and magnetic beads allowed recovery of a high percentage of Gal-DKO cells, for the production of Gal-DKO transgenic pigs.