49 IMPROVED PORCINE CLONING EFFICIENCY WITH CELLS CULTURED FOR SEVERAL GENERATIONS AFTER A SINGLE TREATMENT WITH XENOPUS EGG EXTRACT
Y. Liu A , O. Østrup C , J. Li A , G. Vajta A , P. M. Kragh A , S. Purup B , R. Li A and H. Callesen AA Dept. of Genet. and Biotech., Faculty of Agricultural Sciences, Aarhus University, Aarhus, Denmark;
B Dept. of Anim. Health and Biosci., Faculty of Agricultural Sciences, Aarhus University, Aarhus, Denmark;
C Dept. of Anim. Vet. Basic Sci., KU-LIFE, University of Copenhagen, Copenhagen, Denmark
Reproduction, Fertility and Development 23(1) 130-131 https://doi.org/10.1071/RDv23n1Ab49
Published: 7 December 2010
Abstract
Extracts from eggs of Xenopus laevis frogs can induce nuclear remodelling or increase transcriptional reprogramming in somatic cells. However, it is not known if this effect is passed on from one cell generation to another, or how it affects somatic cell nuclear transfer in porcine cells. This study aimed to investigate the effect of extract-treated cells over several generations on porcine cloning. Extracts were prepared from 2 frogs (B1 and B2) by the same protocol (Higa et al. 2006 Methods 39, 284–290). Fetal fibroblasts grown on poly-L-lysine coated coverslips were permeabilized by digitonin (7 μg mL–1, 2 min, 4°C) and incubated with 1 extract batch at 37°C for 30 min. After resealing the membrane in DMEM supplemented with 2 mM CaCl2 at 37°C for 2 h, the remaining cells were cultured in ES medium (Vejlsted et al. 2005 Mol. Reprod. Dev. 70, 445–454) for 7 to 8 days when they formed colonies. The colonies were trypsinized and divided onto 2 coverslips for subculture, defined as Experimental Passage 1 (XP1). New subcultures were made every 7 to 8 days when 70 to 80% clusters become colonies until XP15. Colonies from XP3, 8 and 15 were isolated and trypsinized before being used in handmade cloning. Nontreated cells grown in DMEM were used as controls (no colony formation was observed). On each cloning day, cells from different XP number and controls were used. Rates of cleavage (Day 2) and blastocyst development (Day 6) were analysed with chi-square test (SAS version 9.2, SAS Institute Inc., Cary, NC, USA). Results are summarised in Table 1. No difference was observed in cleavage rate between groups. Blastocyst rates of all XP colony cells were significantly higher than their controls. For the same XP number and their controls, blastocyst rates were similar between the colony cells from the 2 extract batches, and there was no difference between their controls, either. In conclusion, the cloning efficiency in porcine cells could be increased with extract-treated cells used for several generations, and this effect was present at XP3, 8, and 15.
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