L. F. S. Beebe

Production of homozygous α-1,3-galactosyltransferase knockout pigs by breeding and somatic cell nuclear transfer

Abstract:  We report here our experience regarding the production of double or homozygous Gal knockout (Gal KO) pigs by breeding and somatic cell nuclear transfer (SCNT). Large White × Landrace female heterozygous Gal KO founders produced using SCNT were mated with Hampshire or Duroc males to produce a F1 generation. F1 heterozygous pigs were then bred to half‐sibs to produce a F2 generation which contained Gal KO pigs. To determine the viability of mating Gal KO pigs with each other, one female F2 Gal KO pig was bred to a half‐sib and subsequently a full‐sib Gal KO. F1 and F2 heterozygous females were also mated to F2 Gal KO males. All three types of matings produced Gal KO pigs. To produce Gal KO pigs by SCNT, heterozygous F1s were bred together and F2 fetuses were harvested to establish primary cultures of Gal KO fetal fibroblasts. Gal KO embryos were transferred to five recipients, one of which became pregnant and had a litter of four piglets. Together our results demonstrate that Gal KO pigs can be produced by breeding with each other and by SCNT using Gal KO fetal fibroblasts.

Piglets produced from in vivo blastocysts vitrified using the Cryologic Vitrification Method (solid surface vitrification) and a sealed storage container.

The objective was to develop a simple successful porcine cryopreservation protocol that prevented contact between embryos and liquid nitrogen, avoiding potential contamination risks. In vivo-derived blastocysts were collected surgically from donor pigs, and two porcine embryo vitrification protocols (one used centrifugation to polarize intracytoplasmic lipids, whereas the other did not) were compared using the Cryologic Vitrification Method (CVM), which used solid surface vitrification. The CVM allowed embryos to be vitrified, without any contact between embryos and liquid nitrogen. Both protocols resulted in similar in vitro survival rates (90% and 94%) and cell number (89 ± 5 and 99 ± 5) after 48 h in vitro culture of vitrified and warmed blastocysts. The protocol that did not use centrifugation was selected for continued use. To protect vitrified embryos from contact with liquid nitrogen and potential contamination during storage, a sealed outer container was developed. Use of this sealed outer container did not affect in vitro survival of cryopreserved blastocysts. In vivo blastocysts (n = 151) were collected, vitrified, and stored using the selected protocol and sealed container. These embryos were subsequently warmed and transferred to six recipients; five became pregnant and farrowed a total of 26 piglets. This embryo vitrification method allowed porcine embryos to be successfully vitrified and stored without any contact with liquid nitrogen.

Development of an Improved Porcine Embryo Culture Medium for Cloning, Transgenesis and Embryonic Stem Cell Isolation

Work in our laboratory for more than two decades has focussed on the production of genetically modified pigs for xeno transplantion research. More recent work has focussed on the isolation of porcine embryonic stem cells to facilitate this as well as and other research applications. Central to this research has been the production of in vitro Produced (IVP) embryos. These embryos are produced using a twostep culture system based on NCSU23. This culture system which was developed by modifying energy substrate availability and concentrations and by adding non-essential and essential amino acids in a sequential manner. As a result of this work we have developed a culture system that better suits the changing metabolic needs of the pig embryo and produces embryos with relatively high developmental competence compared to the original formulation. These embryos can be used for a range of research applications including the isolation of embryonic stem cells.

33 USE OF ADULT MESENCHYMAL STEM CELLS FOR CLONING PIGS

Current cloning efficiencies are relatively low and there is evidence to suggest that a less differentiated cell type can increase these (Jaenisch et al. 2002 Cloning and Stem Cells 4, 389-396). As part of an ongoing study that aims to develop cloning as a breeding tool for the pig industry, we examined whether mesenchymal stem cells (MSCs) isolated from bone marrow (Experiment 1) and blood (Experiment 2) could increase cloning efficiencies compared with fibroblasts isolated from ear tissue of live animals. MSCs were isolated from the femurs and blood of two pigs over a Histopaque-1077 density gradient. Cells were seeded and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics/antimycotics. Blood MSCs were plated onto fibronectin-coated dishes (Faast and Nottle, Proc. Australian Health Med. Res. Congress 2004, abst. 1272). Cells from passages 2-4 were used for nuclear transfer by means of a fusion before being subjected to the activation protocol described previously (Harrison et al. 2004 Cloning and Stem Cells 6, 327-331). Embryos were cultured in a modified NCSU23 for 7 days. In Experiment 1 there was no difference in the number of fused embryos that cleaved in the bone marrow MSC and fibroblast groups (374/447; 84% vs. 370/446; 83%, respectively; 9 replicates). The number of embryos that developed to the blastocyst stage by day 7 was significantly higher in the bone marrow MSC group compared with the fibroblast group (136/447; 30% vs. 79/446; 18%, respectively, Chi square test; P < 0.01). In Experiment 2 there was no difference in the percentage of embryos that cleaved in the blood MSC and fibroblast groups (287/375; 77% vs. 275/347; 79%, respectively; 8 replicates). The number of embryos that developed to the blastocyst stage by day 7 was also similar between the blood MSC and fibroblast groups (67/375; 18% vs. 63/347; 18%, respectively). In conclusion, our results suggest that bone marrow MSCs may be a more efficient cell type, compared with fibroblasts, for the cloning of live animals.