40 STREPTOLYSIN O PERMEABILIZED AND MITOTIC EXTRACT TREATED GOAT FETAL FIBROBLASTS FOR USE IN NUCLEAR TRANSFER

Abstract

Nuclear transfer (NT) technology has been limited by low rates of embryonic development, high pregnancy loss and low survival of cloned offspring. A major cause is thought to be incomplete or incorrect reprogramming of the somatic cell nucleus. An alternative NT method involves inducing nuclear envelope breakdown (NEBD) and chromatin condensation (CC) with a mitotic cell extract prior to NT. In this study, caprine fetal fibroblasts were permeabilized with streptolysin O (SLO) and NEBD and CC induced with a mitotic extract prior to NT. Reprogramming was indirectly assessed by measuring DNA methylation in early NT embryos. Permeabilization and resealing of fibroblasts were assessed by propidium iodide (PI) uptake and CC was monitored by staining with Hoechst 33342. All data for percent of cells staining was analyzed with one way ANOVA with significance at P ≤ .05. Cells were treated with 0, 150, 300, or 450 U mL^–1 SLO for 15, 40, 65, or 90 min. A SLO concentration of 300 U mL^–1 for 40 min produced the most efficient permeabilization. Permeabilized cells were treated with extracts from 2, 4 or 6 × 10⁶ mitotic cells for 30, 45 or 60 min. There was no difference in percent of cells with CC between treatments (45–67%) but a higher percentage of treated cells had CC than nontreated cells. Permeabilized and treated cells were resealed by addition of 0, 2.0, 2.5, or 3.0 mm CaCl₂ to the culture medium and incubated for 60, 90, or 120 min. Addition of 2.5 mm CaCl₂ and incubation for 60 min produced the most efficient resealing. Caprine embryos were produced by NT using oocytes from abattoir and superstimulated ovaries. Fibroblasts were permeabilized by incubation with 300 U mL^–1 of SLO at 37°C for 40 min in HBSS with 10 mm dithiothreitol. Fibroblasts were then incubated with mitotic extract for 30 min at 37°C, then cultured in DMEM supplemented with 10% FBS and 2.5 mm CaCl₂ for 1 h. Nuclear transfer was performed using untreated donor cells and SLO and mitotic extract treated (SLOT) fibroblasts from the same fetal cell line. All donor cells were at passage 5 or less and contact inhibited by confluence prior to treatment (SLOT) or fusion (NT). NT with untreated cells produced 21 (8%) 2–4 cell and 10 (4%) 4–8 cell embryos. NT with SLOT cells produced 23 (9%) 2–4 cell and 9 (4%) 4–8 cell embryos. In a separate experiment, IVF embryos were produced from abattoir sourced ooctyes for DNA methylation analysis. Embryos were fixed, permeabilized, and immunostained with an antibody against 5-methylcytosine and observed with a confocal laser scanning microscope. Total DNA methylation in individual nuclei of NT and IVF embryos is being determined by image analysis with Image J software.