Reproductive Technology

Red Deer Cloned from Antler Stem Cells and Their Differentiated Progeny

Abstract
The significance of donor cell differentiation status for successful cloning by somatic cell nuclear transfer (SCNT) is unclear. Here we cloned a new species, red deer (Cervus elaphus), from multipotent antler stem cells and their differentiated progeny. Cultured donor cell lines from male antlerogenic periosteum (AP) were left undifferentiated or chemically induced to initiate osteogenesis or adipogenesis. Based on their morphology and marker gene expression profile, donor cells were classified as undifferentiated AP cells, presumptive osteoblasts or adipocytes. Adipocytes up-regulated adipogenic markers procollagen type I alpha2 (COL1A2), peroxisome proliferator-activated receptor gamma2 (PPARG) and gylceraldehyde-3-phosphate dehydrogenase (GAPDH) and down-regulated antlerogenic transcripts POU-domain class 5 transcription factor (POU5F1) and parathyroid hormone (PTH)-like hormone (PTHLH). Despite differences prior to NT, transcript abundance of donor specific markers COL1A2, PPARG, GAPDH and POU5F1 did not differ significantly in cloned blastocysts (P = 0.10, 0.50, 0.61 and 0.16, respectively). However, donor cell and blastocyst expression levels were completely different for most genes analyzed, indicating their successful reprogramming. The type of donor cell used for NT (AP, bone and fat cells), had no effect on in vitro development to blastocysts (93/248 = 38% vs. 32/73 = 44% vs. 59/183 = 32%, respectively). Likewise, development to weaning was not significantly different between the three cell types (2/46 = 4% vs. 2/7 = 29% vs. 4/31 = 13%, for AP vs. bone vs. fat, respectively). Microsatellite DNA analysis confirmed that the eight cloned red deer calves were genetically identical to the cells used for NT.

Key words: Embryo • Assisted Reproductive Technology • Pregnancy • Developmental biology • Early development