70 PRODUCTION OF CLONES BY FIBROBLAST NUCLEAR TRANSFER FROM AN X-AUTOSOME TRANSLOCATION CARRIER COW

Abstract

Poor reproductive outcome associated with chromosome anomalies is well documented in humans and domestic animals. In cattle, female carriers of Robertsonian and X-autosome translocations tend to be repeat breeders, probably due to synaptic difficulties during meiotic prophase of gametogenesis. Although viable offspring have been obtained through somatic cell nuclear transfer (NT) using adult or fetal cells from normal animals in various species, there have been no reports to date on the application of this technology to translocation carriers. Since NT circumvents meiotic problems encountered by translocation carriers, we used this approach to generate cloned embryos, fetuses and calves from a subfertile Limousin-Jersey crossbred cow previously identified as a carrier of an X-autosome translocation. Primary cultures were established from ear skin biopsies, and used at the 5th or 6th passage for NT. Recipient oocytes were enucleated at 19 h post-maturation (hpm), fused with individual fibroblasts by a single electrical pulse (1.4 KV cm⁻¹, 40 μs) and activated at 24 hpm with a combination of ionomycin (5 μM, 5 min) and cycloheximide (10 μg mL⁻¹). Reconstructed eggs were cultured in SOF at 39°C in a humidified low oxygen atmosphere. In 33 runs involving 2470 oocyte-donor cell complexes, cleavage and blastocyst rates were 88% (2173/2470) and 36% (889/2470), respectively. Two or three blastocysts (Day 7 ± 1) were transferred into each recipient, previously synchronized with a combination of CIDR, GnRH and PGF₂α. Ultrasonography was performed at Days 28 to 60 and at Days 90 and 150. Pregnancy was confirmed on Day 28 in 10 of a total of 22 recipients, 2 of which were later found to be carrying twin fetuses. Of 60 embryos transferred, 11 (18.3% of embryos) survived to Day 42, 6 (10%) to Day 60, and 4 (6.6%) to Day 90. A Day-94 fetus was surgically retrieved to examine the synaptic pattern of meiocytes in fetal ovaries. The fetus and internal organs were normal in appearance, and of normal size (16.5 cm C-R length). The X-autosome translocation was confirmed in blood cultures, and synaptic anomalies involving chromosome 23 and the X chromosome were detected in fetal ovaries. Another clone was delivered by C-section at 276 days but died within 1 h of delivery, while one singleton pregnancy is still ongoing at >200 days. These results demonstrate that NT can be used to produce embryos, fetuses and offspring from an X-autosome translocation carrier, with the potential to facilitate study of synaptic behaviour of female germ cells, and X-inactivation in different cell lineages of cloned blastocysts, and to generate individuals with otherwise poor reproductive prospects. [Supported by NSERC, Canada and OMAFRA]