Cloning Missy: Obtaining Multiple Offspring of a Specific Canine Genotype by Somatic Cell Nuclear Transfer

Abstract

The present study was undertaken to evaluate two activation methods for somatic cell nuclear transfer (SCNT), namely, fusion and simultaneous activation (FSA, fusion medium contains calcium), versus fusion followed by chemical activation (F+CA, fusion medium does not contain calcium), and to evaluate the effects of parity of recipient dogs on the success of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells collected from an 11-year-old female dog named Missy. In the FSA method, oocytes were fused and activated at the same time using two DC pulses of 1.75 kV/cm for 15 microsec. In the F+CA method, oocytes were fused with two DC pulses of 1.75 kV/cm for 15 microsec, and then activated 1 h after fusion by 10 microM calcium ionophore for 4 m and cultured for 4 h in 1.9 mM 6-dimethylaminopurine for postactivation. Activation method had a significant impact on the production efficiency of cloned dogs. There was a significant difference in full-term pregnancy rate and percentage of live puppies between the two methods (6.3% and 38.5% for FSA and F+CA, respectively). In our study, four out of five live offspring produced by F+CA survived versus FSA, which did not result in any surviving puppies. Overall, as few as 14 dogs and 54 reconstructed embryos were needed to produce a cloned puppy. In addition, the parity of recipient bitches had no effect on the success of SCNT in canine species. Both the nullipara and multipara bitches produced live puppies following SCNT-ET.

Altered Cell Cycle Gene Expression and Apoptosis in Post-Implantation Dog Parthenotes

“…Pregnancy was monitored ultrasonographically after the initial confirmation [30], [31], [32], [37]. The conceptuses derived from the AI group (control) and parthenogenetically activated embryos (PA group) were obtained from recipients on days 28, 30 or 32 of pregnancy, and were analyzed for their size of sac, gross external morphology and weight.…”

Establishment of a canine model of human type 2 diabetes mellitus by overexpressing phosphoenolypyruvate carboxykinase

“…During heat, a blood sample (2 ml) was collected daily at the same time by cephalic venipuncture and serum progesterone levels were assayed using a Cobas E411 (Roche Diagnostics, Indianapolis, IN, USA). Oocyte retrieval and cloned embryo transfer were performed using general surgical procedures as described in our previous study (13). Briefly, the oviducts were flushed upward with TCM-199 (Invitrogen Life Technologies) from oocyte donor dogs under surgical procedures.…”

“…A number of recent studies reported successful generation of cloned small-breed (10), medium-sized (11) and large-breed dogs (12). Furthermore, another group described the production of one dog cloned from somatic cells that had been cryopreserved for a long period of time using in vivo oocyte collection protocols (13).…”

Species‐specific Challenges in Dog Cloning

“…In addition, activation protocols fusion followed by chemical activation showed higher full‐term pregnancy rate and birth rate of live puppies than those of fusion and simultaneous activation (Hossein et al. ).…”