Bovine nuclear transfer embryo development using cells derived from a cloned fetus

Area of Science:

• Reproductive biology
• Cell biology
• Genetics

Background:

• Nuclear transfer (NT) is a technique used to create cloned embryos and fetuses.
• The potential of using fibroblasts from cloned fetuses as donor cells for subsequent NT is largely unknown.
• Assessing the normality and potential abnormalities in successive generations of clones is crucial.

Purpose of the Study:

• To evaluate the efficacy of bovine fibroblast cells derived from a cloned fetus as donor cells for nuclear transfer.
• To compare the developmental potential of early and late passage cloned fetal fibroblasts in vitro and in vivo.
• To determine if cloned fetuses can be recloned to produce subsequent identical clones.

Main Methods:

• Generated a long-term bovine fibroblast cell line from a cloned fetus.
• Compared early passage (P2) and late passage (P18) cells as NT donor cells.
• Assessed in vitro blastocyst development rates and in vivo pregnancy rates after embryo transfer.
• Analyzed cell cycle distribution (G0/G1) under different culture conditions.

Main Results:

• No significant difference in blastocyst development rates between P2 (35.4%) and P18 (44.6%) cells.
• Late passage cells showed a higher proportion in G0/G1 phase under both serum-fed and starved conditions.
• Equivalent day 30 pregnancy rates were observed for both groups (P2: 2/19, P18: 2/13).
• Fetuses developed to day 56 but none survived to term, suggesting potential issues with gestation or sample size.

Conclusions:

• Fibroblast cells derived from cloned fetuses are capable of supporting in vitro and in vivo development after nuclear transfer.
• The longevity of these cell lines allows for extended periods for genetic manipulation and selection of transfected cells.
• The study provides encouraging evidence that cloned fetuses can be recloned, though further research is needed to confirm live offspring production and identify failure causes.