MİTHAT EVECEN, SERHAT PABUCCUOĞLU, KAMBER DEMİR, Selin YAĞCIOĞLU, Ayşe CAN, Ezgi ERTÜRK, Asiye İzem SANDA, RAMAZAN ARICI, GÜL BAKIRER ÖZTÜRK, KEMAL AK, Sema BİRLER

Somatic Cloning in Cats Using MI or MII Oocytes [1]

Somatik klonlama yoluyla hayvan üretimi, üstün değerdeki bireylerin korunması, savunmasız ve tehlike altında bulunan türlerin korunması ile transgenik hayvanların çoğaltılmasına hizmet eder. Çalışmanın materyalini 167 adet MI ve 219 adet MII dönemdeki oosit oluşturdu. Polar cisimciklerin (MII) ve kromatin setlerin (MI ve MII) enükleasyonu, 44 saatlik in vitro olgunlaştırma periyodunun ardından gerçekleştirildi. Nükleer transfer amacıyla siklik dönemlerdeki granüloza hücreleri kullanıldı. Oositsomatik hücre komplekslerinde füzyon işlemi, DC akımın sağlandığı 2.0 kV/cm 60 µs, 0.1s ara (2x), BTX Electrocell Manipulator 200 (BTX, San Diego, CA, USA) ile gerçekleştirildi. Aktivaston işlemi için ise, 1.0 kV/cm 20µs DC akım 0.1s ara (2x) kullanıldı ve ardından 2 mM 6-DMAP (6-dimethylaminopurine) içerisine alınarak, 38°C'lik sıcaklık ve %5 CO2, %5 O2 ve %90 N2 gaz karışımının sağlandığı inkübatörde 4 saat süresince kültüre edildi. Somatik hücrelerin nakledildiği klon embriyolar daha sonra aynı inkübatör koşullarında %0.4 BSA katkılı mSOF medyumu içerisinde 8 gün boyunca in vitro kültüre bırakıldılar. Ardından klon embriyolar, embriyonik hücre sayılarının tespiti amacıyla Hoechst 33342 (5 ?g/mL) içeren HSOF medyumu içerisine alındı ve ultraviyole küplü floresan mikroskobunda değerlendirildi. Sonuçta, füzyon (%66.66-21.55) ve yarıklanma oranlarının (%15.75-11.11) MII dönem oositleri lehine önemli derecede üstün olduğu belirlendi (P

Kedilerde MI ve MII Oositleri Kullanilarak Somatik Klonlama

Animal production via SCNT provides a unique tool for protection of valuable individuals, conservation of vulnerable and endangered species and production of transgenic animals. A total of 167 MI and 219 MII stage oocytes were used as the material of the study. The oocytes were enucleated at 44 h after in vitro maturation by aspiration of the polar body and the MI or MII plates. Cycling granulosa cells were used for nuclear transfer. Cell fusion was induced with DC pulses of 2.0 kV/cm 60µs, 0.1s apart (2x) delivered by a BTX Electrocell Manipulator 200 (BTX, San Diego, CA, USA). After fusion, the embryos were activated by 1.0 kV/cm 20µs DC pulses 0.1s apart (2x) followed by 2 mM 6-DMAP (6-dimethylaminopurine) incubation in culture medium for 4 h in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 at 38°C. The somatic cell transferred embryos were cultured for 8 days in mSOF medium supplemented with 0.4% BSA in a humidified 5% CO2, 5% O2, and 90% N2 atmosphere at 38°C. After in vitro culture period, all embryos transferred to HSOF containing Hoechst 33342 (5 ?g/mL) and the cell numbers were counted under ultraviolet light using a fluorescent microscope. The fusion (66.66 vs 21.55%) and cleavage rates (15.75 vs 11.11%) were significantly higher in MII stage oocytes than MI stage oocytes (P<0.02). While SCNT embryos were developed to morula stage in MII group (14; 9.58%), all the cleaved embryos were arrested at the 2-4 cell stage in MI group. None of the embryos was developed to blastocyst stage in both groups.

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Pope CE: Embryo technology in conservation efforts for endangered felids. Theriogenology, 53, 163-174, 2000. DOI: 10.1016/S0093-691X (99)00249-6
Gomez MC, Pope CE, Giraldo A, Lyons LA, Harris RF, King AL, Cole A, Godke RA, Dresser BL: Birth of African Wildcat cloned kittens born from domestic cats. Cloning Stem Cells, 6, 247-258, 2004. DOI: 10.1089/ clo.2004.6.247
Gomez MC, Jenkins JA, Giraldo A, Harris RF, King A, Pope CA: Nuclear transfer of synchronized African wild cat somatic cells into enucleated domestic cat oocytes. Biol Reprod, 69, 1032-1041, 2003. DOI: 1095/biolreprod.102.014449
Biller DS, DiBartola SP, Eaton KA, Pflueger S, Wellman ML, Radin MJ: Inheritance of polycystic kidney disease in Persian cats. J Hered, , 1-5, 1986.
Baker HJ, Walkley SU, Rattazzi MC, Singer HS, Watson HL, Wood PA: Feline gangliosidoses as models of human lysosomal storage diseases. Prog Clin Biol Res, 94, 203-212, 1982.
Murphy WJ, Sun S, Chen ZQ, Pecon-Slattery J, O'Brien SJ: Extensive conservation of sex chromosome organization between cat and human revealed by parallel radiation hybrid mapping. Genome Res, 9, 1223-1230, DOI: 10.1101/gr.9.12.1223
Wilmut I, Schnieke, AE, McWhir J, Kind AJ, Campbell KHS: Viable offspring derived from fetal and adult mammalian cells. Nature, 385, 810- , 1997. DOI: 10.1038/386200a0
Birler S, Pabuccuoğlu S, Demir K, Cirit U, Karaman E, Bacınoğlu S, Özdaş ÖB, Evecen M, Alkan S, Baran A, Bakırer G, Özcan C, Koçak Ö, Kılıçarslan R, Kaşıkçı G, Toydemir S, Dinç H, Koban E, Togan İ, İleri İK, Ak K: Production of cloned lambs: Transfer of early cleavage stage embryos to final recipients. J Fac Vet Med Ist Univ, 36, 1-8, 2010.
Li Z, Sun X, Chen J, Liu X, Wisely SM, Zhou Q, Renard JP, Leno GH, Engeldardt JF: Cloned ferrets produced by somatic cell nuclear transfer. Dev Biol, 293, 439-448, 2006. DOI: 10.1016/j.ydbio.2006.02.016
Lee BC, Kim MK, Jang G, Oh HJ, Yuda F, Kim HJ, Shamim, MH, Kim JJ, Kang SK, Schatten G, Hwang WS: Dogs cloned from adult somatic cells. Nature, 436, 641, 2005. DOI: 10.1038/436641a
Galli C, Lagutina, Crotti G, Colleoni, S, Turini P, PonderatoN, Roberto LazzariDG: A cloned foal born to its dam twin. Nature, 424, 635, DOI: 10.1038/424635a
Woods, GL, White KL, Vanderwall DK, Li GP, Aston KI, Bunch TD, Meerdo LN, Pate BJ: A mule cloned from fetal cells by nuclear transfer. Science, 22, 301(5636), 1063, 2003. DOI: 10.1126/science.1086743
Shin T, Kraemer D, Pryor J, Liu L, Rugila J, Howe L, Buck S, Murphy K, Lyons L, Westhusin M: Cell biology: A cat cloned by nuclear transplantation. Nature, 415, 859, 2002. DOI: 10.1038/nature723
Onishi A, Iwamoto M, Akita T, Mikawa S, Takeda K, Awata T, Hanada H, Perry AC: Pig cloning by microinjection of fetal fibroblast nuclei. Science, 289, 1188-1190, 2000. DOI: 10.1126/science.289.5482.1188
Kato Y, Tani T, Tsunoda Y: Cloning of calves from various somatic cell types of male and female adult, newborn and fetal cows. J Reprod Fertil, , 231-237, 2000. DOI: 10.1530/jrf.0.1200231
Baguisi A, Behboodi E, Melican DT, Pollock JS, Destrempes MM, Cammuso C, Williams JL, Nims SD, Porter CA, Midura P, Palacios MJ, Ayres SL, Denniston RS, Hayes ML, Ziomek CA, Meade HM, Godke RA, Gavin WG, Overstrom EW, Echelard Y: Production of goats by somatic cell nuclear transfer. Nat Biotechnol, 17, 456-461, 1999. DOI: 10.1038/8632
Wakayama T, Perry ACF, Zuccotti M, Johnson KR, Yanagimachi R: Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature, 394:369-374, 1998. DOI: 10.1038/28615
Fissore RA, He CL, Woude GFV: Potential role of mitogen activated protein kinase during meiosis resumption in bovine oocytes. Biol Reprod, , 1261-1270, 1996. DOI: 10.1095/ biolreprod55.6.126
Campbell KHS, Loi P, Otaegui P, Wilmut I: Cell cycle co-ordination in embryo cloning by nuclear transfer. Rev Reprod, 1, 40-45, 1996. DOI: 1530/ror.0.0010040
Whitaker M: Control of meiotic arrest. Rev Reprod, 1, 127-135, 1996. DOI: 10.1530/ror.0.0010127
Nurse P: Universal control mechanism regulating onset of M-phase. Nature, 344, 503-508, 1990. DOI: 10.1038/344503a0
Stice SL, Robl JM: Nuclear reprogramming in nuclear transplant rabbit embryos. Biol Reprod, 39, 657-664, 1988. DOI: 10.1095/ biolreprod39.3.657
Campbell KHS, Ritchie W, Wilmut I: Nuclear-cytoplasmic interactions during the first cell cycle of nuclear transfer reconstructed bovine embryos: Implications for deoxyribonucleic acid replication and development. Biol Reprod, 49, 933-942, 1993. DOI: 10.1095/ biolreprod49.5.933
DiBerardino MA, Orr Hoffner N, McKinnell RG: Feeding tadpoles cloned from Rana erythrocyte nuclei. Proc Natl Acad Sci USA, 83, 8231- , 1986.
Miyoshi K, Rzucidlo SJ, Gibbons JR, Arat S, Stice SL: Development of porcine embryos reconstituted with somatic cells and enucleated metaphase I and II oocytes matured in a protein free medium. BMC Dev Biol, 1-12, 2001. DOI: 10.1186/1471-213X-1-12
Demir K, Can A, Ertürk E, Özdemirci S, Karaçam H, Sandal AI, Öztürk GB, Arıcı R, Çınar EM, Birler S, Pabuccuoğlu S, Ak K, Evecen M: Effect of different activation techniques on immature and in vitro matured cat oocytes. Kafkas Univ Vet Fak Derg, 20, 565-570, 2014. DOI: 9775/kvfd.2013.1061
Yin XJ, Lee HS, Kim LH. , Shin HD, Kim NH, Kong IK: Effect of serum starvation on the efficiency of nuclear transfer using odd-eyed white cat fibroblasts. Theriogenology, 67, 816-823, 2007. DOI: 10.1016/j. theriogenology.2006.10.010
Gomez MC, Pope CA, Dresser BL: Nuclear transfer in cats and its application. Theriogenology, 66, 72-81, 1996. DOI: 10.1016/j.theriogenology. 03.017
Keefer CL: Lessons learned from nuclear transfer (cloning). Theriogenology, 69, 48-54, 2008. DOI: 10.1016/j.theriogenology.2007.08.033
Birler S, Pabuccuoğlu S, Demir K, Cirit Ü, Karaman E, Evecen M, Özdaş ÖB, Alkan S: Effects of serum starvation and ionomycine treatments on somatic cell nuclear transfer in sheep. Reprod Dom Anim, (Suppl. 3): 90, 2011 (Abstract). DOI: 10.1111/j.1439-0531.2011.01839.x
Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce de Leon FA, Robl JM: Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science, 280, 1256-1258, 1998. DOI: 1126/science.280.5367.1256
Bochenek M, Katska L, Kania G, Rynska B, Smorag Z, Pienkowski M: Cell cycle analysis of somatic cell lines in the cloning of the domestic cat. Theriogenology, 55, 256, 2001 (Abstract). DOI: 10.1016/S0093-691X (00)00457-X
Farstad W: Current state in biotechnology in canine and feline reproduction. Anim Reprod Sci, 60-61, 375-387, 2000. DOI: 10.1016/S0378- (00)00106-8
Skrzyszowska M, Katska L, Rynska B, Kania G, Smorag Z, Pienkowski M: In vitro developmental competence of domestic cat embryos after somatic cloning: A preliminary report. Theriogenology, 58, 1621, 2002. DOI: 10.1016/S0093-691X(02)01047-6
Robinson JJ: Nutrition and Reproduction. Anim Reprod Sci, 42, 25-34, DOI: 10.1016/0378-4320(96)01526-6