Genomic Stability of Lyophilized Sheep Somatic Cells before and after Nuclear Transfer

Abstract

The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT). Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT.

Freeze-dried spermatozoa: An alternative biobanking option for endangered species

“…In other words, drying spermatozoa is in its infancy, and there is ample margin for improving, starting from the development of media, to radical chances in all the parameters, like freezing temperatures and the vacuum conditions. The finding so far achieved using the current, empirical state of art induces to a cautious optimism (Wakayama and Yanagimachi, 1998;Kusakabe et al, 2001;Hirabayashi et al, 2005;Kusakabe et al, 2008;Loi et al, 2008aLoi et al, , 2008bGianaroli et al, 2012;Iuso et al, 2013) This finding supports the development of low cost, on the shelf genetic biobanks from domestic and also wild endangered species.…”

45 DNA Fragmentation of Epididymal Freeze-Dried Ram Spermatozoa Impairs Embryo Development

“…Most of these chromosome alterations are highly unstable, affecting mitotic chromosome segregation, resulting in chromosome imbalances in the newly produced cells, with subsequent cell death and poor or inhibited embryonic development. Moreover, the induction of DSBs caused by the lyophilization process was also evaluated in sheep lymphocytes [37]. Iuso et al [37] proved that the formation of DSBs in lymphocytes is a consequence of the lyophilization.…”

“…Moreover, the induction of DSBs caused by the lyophilization process was also evaluated in sheep lymphocytes [37]. Iuso et al [37] proved that the formation of DSBs in lymphocytes is a consequence of the lyophilization. In our work, where spermatozoa were used, the levels of DSBs were relatively low (a range between 0.3% and 4.6%), suggesting that sperm DNA structure responds better to freeze-drying (in terms of DSBs).…”

Cloning the Mammoth: A Complicated Task or Just a Dream?

“…The redundancy of DNA repairing machinery is by all means unexpected and surprising. In our recent study (Iuso et al 2013 ) on nuclear transfer of lyophilized cells, we addressed the issue of DNA lesions caused by the freeze-drying process and whether they are repaired after nuclear transfer. DNA damage was indeed observed in dried lymphocytes, but after nuclear transfer the resulting pronucleus was stuffed with foci of active DNA repair (identifi ed with an antibody raised against modifi ed histone recruited at sites of DNA repair (Podhorecka et al 2010 )).…”

“…19.4 Immuno-localization of histone variant gamma H2AX, which is recruited at sites of DNA repair, in lyophilized cells injected into enucleated sheep oocytes. The DNA repairing activity of the oocyte is not diluted even in case four somatic nuclei are injected (4 NT; the nucleus in the upper part of the oocyte has divided, an event which often occurs in SCNT), suggesting DNA repairing activity of the oocyte is highly redundant; hence it might turn out to be a mighty allied in a mammoth cloning project (Iuso et al 2013 ) Hence, hybrid mammoth/elephant embryos will be allowed to develop to blastocyst stage using the culture conditions most appropriate for elephant embryo. What these culture conditions might be is still a big question mark since any of the in vitro techniques related to elephant oocytes and embryos have never been attempted, or at least was not reported on.…”