Andrew Boquest

Publications (2)5.32 Total impact

• Article: A comparison of two in vitro maturation media for use with adult porcine oocytes for adult somatic cell nuclear transfer.

  Luke Beebe, Stephen McIlfatrick, Christopher Grupen, Andrew Boquest, Sharon Harrison, Renate Faast, Rodney Ashman, James Wengle, Hamish Hamilton, Mark Nottle
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  ABSTRACT: Two media used to mature adult porcine oocytes for somatic cell nuclear transfer were compared. In the first experiment, parthenogenetic embryos were produced using a maturation medium used by us previously to clone pigs (OMM199) and that described by Kühholzer et al. (2001) to transport oocytes overnight (BOMED). There was no difference in maturation rates between the two different media. However, BOMED medium increased the percentage of parthenogenetic embryos that developed to the blastocyst stage compared with OMM199 (49% vs. 29%, respectively). In a second experiment, BOMED medium increased the percentage of SCNT embryos that developed to the blastocyst stage compared with OMM199 (22% vs. 8%, respectively). The efficiency of our cloning protocol using adult oocytes matured in BOMED medium was then determined by transferring SCNT embryos reconstructed using adult fibroblasts to synchronized recipients. Primary cultures of adult fibroblasts were obtained from two adult male pigs and used for SCNT (passages 2-4). Between 82 and 146 fused couplets were transferred to seven recipients synchronized 1 day behind the embryos. Five recipients (71% pregnancy rate) subsequently farrowed a total of 23 piglets (4.4 average litter size). Overall efficiencies (liveborn/embryos transferred) were 3.2% for all transfers and 4.3% for animals that gave birth.
  Cloning and Stem Cells 02/2007; 9(4):564-70. · 2.66 Impact Factor
• Article: An efficient method for producing alpha(1,3)-galactosyltransferase gene knockout pigs.

  Sharon Harrison, Andrew Boquest, Christopher Grupen, Renate Faast, Angelo Guildolin, Christopher Giannakis, Lesley Crocker, Stephen McIlfatrick, Rodney Ashman, James Wengle, Ian Lyons, Paul Tolstoshev, Peter Cowan, Allan Robins, Philip O'Connell, Anthony J F D'Apice, Mark Nottle
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  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.
  Cloning and Stem Cells 02/2004; 6(4):327-31. · 2.66 Impact Factor