Can a chemical treatment help cloned rat embryos develop beyond their usual early stopping point?

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Although somatic cell nuclear transfer (SCNT) had produced cloned animals in many species, only one previous report had described successful production of cloned rats. Earlier researchers studying reconstructed rat embryos found that they did not progress beyond the 2-cell stage, attributing this to inadequate formation of the mitotic assembly and nuclear organisation, or improper transcription of cytoskeleton genes.

The study team had previously improved the proportion of reconstructed rat embryos forming two pronuclei by treating them with the proteasome inhibitor MG132, but no embryos developed beyond the 2-cell stage (Nakajima et al., 2008, Cloning and Stem Cells 10, 461–468). Treatment with the histone deacetylase inhibitor trichostatin A (TSA) had been shown to dramatically improve mouse cloning efficiency, although its precise effect in SCNT remained unclear. Its applicability to rats had not been tested. This study aimed to determine whether TSA treatment was also effective in rat SCNT.

In this controlled laboratory experiment, the researchers collected rat oocytes and removed their spindles in medium containing MG132, following their previous reports. After enucleation, they injected cumulus-cell nuclei into the oocytes and cultured the reconstructed embryos in medium containing 0 nM TSA (control), 5 nM, 50 nM, or 500 nM TSA. Each treatment group used more than 100 embryos. They evaluated pronuclear formation, development to the 2-cell stage, and blastocyst formation.

TSA treatment affected neither the proportion of embryos forming pronuclei nor development to the 2-cell stage. However, 50 and 500 nM TSA—higher than the 5 nM concentration usually used for mouse SCNT—enabled reconstructed rat embryos to reach the blastocyst stage, at a low rate of 1.4–2.2%. No embryos reached the blastocyst stage in either the 5 nM group or the control group.

The researchers concluded that TSA treatment also appears effective in rat SCNT and that these findings would be useful for improving the rat SCNT protocol.

The work was supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan through a Grant-in-Aid for Matching Fund Subsidy for Private Universities to J. I. and N. K.