Abstract
Successful cloning of animals using somatic cell nuclear transfer requires undamaged nuclear DNA from desired donor cell types. In vitro culture of cells is one way of ensuring nuclear integrity. The goal of this study was to evaluate the limits of postmortem cell survival/culture in refrigerated goat ear skin tissues which could be used for long-term storage and cloning of animals in future. To achieve this, 60 explants from 6 different goats were cultured after 0, 3, 6, 9, 13, 16, 20, 23, 27, 30, 33, 37, and 41 d postmortem and observed under inverted microscope for outgrowth of fibroblast-like cells, after 10–12 d of culture. Explants from all time points including 19% from 41-dpm tissues exhibited outgrowth. However, the percentage of outgrowth positive explants, as well as culture confluence, reduced with increasing postmortem time interval. Cell cultures established from primary outgrowth of 41-dpm tissues when compared for their growth profile with similarly obtained 0-dpm cultures revealed similar growth curve and cell morphology. Cytogenetic analysis of 41-dpm tissue-derived cell populations revealed a normal female karyotype with 60 XX homologous chromosomes indicating genetic stability of the cell population. In conclusion, these results show that refrigerated skin tissue remains alive for more than a month and that the cells derived from such tissues are normal and can be cryopreserved for long-term storage and future cloning of animals with desired genetics.
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References
Erker L, Azuma H, Lee AY, Guo C, Orloff S, Eaton L, Benedetti E, Jensen B, Finegold M, Willenbring H, Grompe M (2010) Therapeutic liver reconstitution with murine cells isolated long after death. Gastroenterology 139:1019–1029
Hoshino Y, Hayashi N, Taniguchi S, Kobayashi N, Sakai K, Otani T, Iritani A, Saeki K (2009) Resurrection of a bull by cloning from organs frozen without cryoprotectant in a −80 degrees C freezer for a decade. PLoS ONE 4:e4142
Jaszczak K, Czlonkowska M, Guszkiewicz A, Parada R (1991) Cytogenetic analysis of experimental interspecies goat-sheep chimera. J Hered 82:244–245
Jaszczak K, Parada R, Guszkiewicz A (1999) Cytogenetic study of some tissues and age-related changes in cell proportions in a goat-sheep chimera. Cytogenet Cell Genet 84:55–57
Latil M, Rocheteau P, Chatre L, Sanulli S, Memet S, Ricchetti M, Tajbakhsh S, Chretien F (2012) Skeletal muscle stem cells adopt a dormant cell state post mortem and retain regenerative capacity. Nat Commun 3:903
Li XC, Yue H, Li CY, He XH, Zhao QJ, Ma YH, Guan WJ, Ma JZ (2009) Establishment and characterization of a fibroblast cell line derived from Jining Black Grey goat for genetic conservation. Small Ruminant Res 87:17–26
Loi P, Ptak G, Barboni B, Fulka J Jr, Cappai P, Clinton M (2001) Genetic rescue of an endangered mammal by cross-species nuclear transfer using post-mortem somatic cells. Nat Biotechnol 19:962–964
Mastromonaco GF, Perrault SD, Betts DH, King WA (2006) Role of chromosome stability and telomere length in the production of viable cell lines for somatic cell nuclear transfer. BMC Dev Biol 6:41
Meisner LF, Johnson JA (2008) Protocols for cytogenetic studies of human embryonic stem cells. Methods 45:133–141
O’Brien SJ, Menninger JC, Nash WG (2006) Atlas of mammalian chromosomes: 653–655.
Ogura A, Inoue K, Wakayama T (2013) Recent advancements in cloning by somatic cell nuclear transfer. Philos Trans R Soc Lond B Biol Sci 368:20110329
Palmer TD, Schwartz PH, Taupin P, Kaspar B, Stein SA, Gage FH (2001) Cell culture. Progenitor cells from human brain after death. Nature 411:42–43
Silvestre MA, Saeed AM, Cervera RP, Escriba MJ, Garcia-Ximenez F (2003) Rabbit and pig ear skin sample cryobanking: effects of storage time and temperature of the whole ear extirpated immediately after death. Theriogenology 59:1469–1477
Silvestre MA, Sanchez JP, Gomez EA (2004) Vitrification of goat, sheep, and cattle skin samples from whole ear extirpated after death and maintained at different storage times and temperatures. Cryobiology 49:221–229
Singh M, Sharma AK (2011) Outgrowth of fibroblast cells from goat skin explants in three different culture media and the establishment of cell lines. In Vitro Cell Dev Biol Anim 47:83–88
Singh M, Ma X, Amoah E, Kannan G (2011) In vitro culture of fibroblast-like cells from postmortem skin of Katahdin sheep stored at 4 degrees C for different time intervals. In Vitro Cell Dev Biol Anim 47:290–293
Singh M, Ma X, Sharma A (2012) Effect of postmortem time interval on in vitro culture potential of goat skin tissues stored at room temperature. In Vitro Cell Dev Biol Anim 48:478–482
Strober W (2001) Trypan blue exclusion test of cell viability. Curr Protoc Immunol Appendix 3: Appendix 3B
Viel JJ, McManus DQ, Cady C, Evans MS, Brewer GJ (2001) Temperature and time interval for culture of postmortem neurons from adult rat cortex. J Neurosci Res 64:311–321
Wakayama S, Ohta H, Hikichi T, Mizutani E, Iwaki T, Kanagawa O, Wakayama T (2008) Production of healthy cloned mice from bodies frozen at −20 degrees C for 16 years. Proc Natl Acad Sci U S A 105:17318–17322
Wikipedia. List of animals that have been cloned. http://en.wikipedia.org/wiki/List_of_Animals_That_Have_Been_Cloned
Xu Y, Kimura K, Matsumoto N, Ide C (2003) Isolation of neural stem cells from the forebrain of deceased early postnatal and adult rats with protracted post-mortem intervals. J Neurosci Res 74:533–540
Acknowledgments
Technical assistance by Xiaoling Ma is thankfully acknowledged. Part of this work was presented as an oral presentation in 17th Biennial ARD Research Symposium, Gainesville, FL, April 7–10, 2013; Abstract No: 11. This work was partly supported by a USDA-NIFA Capacity Building Grant No: 2011-38821-30910 to MS.
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Okonkwo, C., Singh, M. Recovery of fibroblast-like cells from refrigerated goat skin up to 41 d of animal death. In Vitro Cell.Dev.Biol.-Animal 51, 463–469 (2015). https://doi.org/10.1007/s11626-014-9856-9
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DOI: https://doi.org/10.1007/s11626-014-9856-9