An Efficient Method for Producing α(1,3)-Galactosyltransferase Gene Knockout Pigs

Abstract

We have reported relatively efficient methods for somatic cell nuclear transfer and for knocking out the alpha(1,3)-galactosyltransferase (alpha1,3-GT) gene in porcine fetal fibroblasts using a nonisogenic promoterless construct approach. Here we report the production of alpha1,3-GT gene knockout pigs using these procedures. Seven alpha1,3-GT gene knockout cell clones were identified by long-range PCR from 108 neomycin resistant (neo(R)) colonies, giving a 6.5% targeting efficiency. Three cell clones were used for nuclear transfer. Nuclear transfer was performed using a fusion before activation protocol using in vitro-matured adult oocytes. Between 51 and 110 fused couplets were transferred to 10 recipients synchronized 1 day behind the embryos. Parturition was induced on day 115, and piglets were delivered by caesarean section. Four recipients gave birth to a total of 18 live piglets. All pigs were female, and all three clones resulted in the birth of live pigs. alpha1,3-GT gene knockout pigs were identified by long-range PCR and confirmed by Southern blot analysis. The efficiency (embryos transferred/piglets born) of our cloning protocol was 1.9% for all transfers and 4.6% for animals that gave birth.

Thrombotic Microangiopathy

“…First, these data confirm what was already suspected: The ␣Gal epitope needs to be removed, complement activation needs to be prevented, and pig EC need to be maintained in an antithrombotic state. Already, ␣Gal, the major target of preformed anti-pig antibody in humans, has been deleted in pigs (12,13). As Shimizu et al comment, the use of ␣Gal knockout pigs has had a significant impact on the severity of renal thrombotic microangiopathy after pig renal xenotransplantation in baboons (14).…”

Xenotransplantation Transgenesis. Are we there yet?

“…The advent of animal cloning with the birth of Dolly [2] changed this however as ESCs were longer needed to perform a knockout. Subsequently we and several others produced cloned pigs [4,5] and then GTKO pigs [6] within a decade of Dolly. This was a remarkable achievement given the number of technologies that had to be developed, from in vitro oocyte maturation [7] to the use of promoter-less constructs to increase targeting efficiencies to workable levels [8] for this to happen.…”

Multipotent Cell Types in Primary Fibroblast Cell Lines Used to Clone Pigs using Somatic Cell Nuclear Transfer

“…Fibroblasts were seeded concurrently at 1-2 × 10 4 cells/ cm 2 into 4 well trays and maintained at confluence for 5-10 days, prior to harvesting for SCNT. The fusion before activation protocol used was essentially as described previously by us [4] with the exception that enucleation was performed using Hoechst 33342. Cumulus oocyte complexes were obtained from adult ovaries and matured in TCM-199 (Invitrogen) supplemented with 5 μg/ml insulin, 10 ng/mL EGF, 0.5 µM cysteamine, 0.2 mM Na-pyruvate, 5 µg/mL FSH, 75 µg/ml penicillin-G, 50 µg/mL streptomycin sulphate and 10% sow follicular fluid in a humidified atmosphere of 5% CO 2 in air.…”

“…We have previously reported the production of cloned pigs using fetal fibroblasts [3], genetically modified (α1,3-galactosyltransferase knockout) fetal fibroblasts [4] and adult fibroblasts isolated from live animals [5]. Based our experience and that of others, new approaches are required to increase these efficiencies, particularly if animal cloning is to be used commercially for breeding purposes.…”