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Cloning rabbits from fetal fibroblasts
Livestock Science 122 (2009) 77–80
Contents lists available at ScienceDirect
Livestock Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i v s c i
Short communication
Cloning rabbits from fetal fibroblasts Shangang Li a,1, Xuejin Chen b,1, Jianjun Shi b, Yi Guo b, Chunguang Yin a, Lixin Du a, Hui Z. Sheng b,⁎ a b
National Center for Molecular Genetics and Breeding of Animal, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, PR China Center for Developmental Biology, Xinhua Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, PR China
a r t i c l e
i n f o
Article history: Received 12 May 2008 Received in revised form 3 July 2008 Accepted 4 July 2008 Keywords: Rabbit Nuclear transfer Fetal fibroblast Reproduction
a b s t r a c t Live rabbits were generated through nuclear transfer using adult cells as nuclear donors. We showed that, in addition to adult cell nuclei, cultured fetal rabbit fibroblasts could support fullterm embryonic development following nuclear transfer to enucleated oocytes. Following nuclear transfer, 24.4% (21/86) of resulting embryos developed to the blastocyst stage, and 289 embryos were transferred to oviducts of 11 recipient mothers, resulting in 6 pregnancies. Three mothers carried the pregnancy to term, and three newborn rabbits were subsequently delivered by caesarian, one of which survived for more than 4 months. DNA analyses confirmed that all 3 rabbits were genetically identical to fetal donor cells. This study demonstrated that rabbits could be cloned from fetal fibroblasts, although at a lower frequency than when using adult somatic cells as nuclear donors. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Somatic cell nuclear transfer (SCNT) offers new opportunities for species production and preservation. The rabbit is the preferred model organism for many biomedical and pharmaceutical studies. Improving the efficiency of rabbit cloning and understanding mechanisms underlying effective cloning are important goals in the field (Mitalipov et al., 1999; Ogura et al., 2000; Dinnyes et al., 2001; Inoue et al., 2002; Yin et al., 2002; Yang et al., 2007). Rabbits have been cloned from nuclei of freshly isolated cumulus cells (Chesne et al., 2002) and adult fibroblasts (Li et al., 2006). Another goal of cloning is to develop efficient methods for introducing genes into rabbits for a variety of applications. In other species, fetal cells are often used for transgenic manipulation (Schnieke et al.,1997; Cibelli et al., 1998; Lai et al., 2002; Ramsoondar et al., 2003). Thus, methods for cloning rabbits from fetal cells would contribute to advancements in rabbit transgenesis as well. However, similar experiments in the rabbit have not yet been reported (Chesne et al., 2006).
⁎ Corresponding author. Center for Developmental Biology, Xinhua Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, 200092, PR China. Tel./fax: +86 21 55570017. E-mail address: [email protected] (H.Z. Sheng). 1 These authors contributed equally to the paper. 1871-1413/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2008.07.019
Here, we present methods for cloning live rabbits from fetal fibroblasts. This will contribute to extending the use of rabbit models by transgenesis. 2. Materials and methods Culture media were purchased from Gibco Invitrogen (Grand Island, NY, USA), unless otherwise noted. Protocols for animal care, SCNT, embryo transfer and Caesarean delivery of rabbits were approved by the Ethics Committee, Xinhua Hospital, Shanghai Jiao Tong University, School of Medicine. 2.1. Preparation of fetal fibroblasts Skin was isolated from the back of a New Zealand White fetus on embryonic day 20 (E20). The tissue was cut into 1 mm cubes, digested with 0.05% trypsin/EDTA for 20 min at 37 °C, transferred into a 25-cm2 culture flask containing 6–8 ml DMEM supplemented with 10% fetal calf serum (FCS, Hyclone Co., Salt Lake City, UT, USA) and cultured at 37 °C in 5% CO2. After approximately 6 days, cells grew out from explants and covered the flask surface. Cells were then treated with 0.05% trypsin/ EDTA and passaged to 3 new flasks (passage 1). The fetal fibroblasts were frozen in liquid nitrogen at passage 3 for future use. To prepare donor cells, 105 fibroblasts from 5–15 passages
78
S. Li et al. / Livestock Science 122 (2009) 77–80
Table 1 A summary in development of nuclear transfer embryos in vivo Sample size
Developmental rate
No. reconstructed No. fused couplets (% from reconstructed) No. cleaved embryos (% from fused) No. transferred embryos No. recipient mothers No. recipients pregnant (% of total recipients) No. recipients delivering (% of total recipients) No. full-term fetuses (% from transferred embryos) No. living fetuses (% from transferred embryos) No. alive at weaning (% from transferred embryos)
618 456 (73.8) 335 (73.5) 289 11 6 (54.5) 3 (27.3) 3 (1.0) 2 (0.7) 1 (0.3)
were cultured to confluence in a 3.5 cm dish and then were in DMEM supplemented with 0.5% FCS for 2–4 days. Cells were harvested with trypsin, resuspended in DMEM with 10% FCS, kept at 4 °C to prevent aggregation. Before nuclear transfer, donor cells were incubated at 37 °C for 10 min, and transferred into micro-drops for microinjection. 2.2. Oocyte collection Adult female White New Zealand rabbits were superovulated as previously published (Li et al., 2006). Oviducts were collected 14 h after hCG injection, flushed with prewarmed RD medium (Carney and Foote, 1991) supplemented with 10% (v/v) FCS and 10 mM Hepes (Hepes balanced RD, hRD). Cumulus cells were removed by exposure to 100 IU/ml hyaluronidase (Sigma chemical Co.) in hRD medium for 10 min and then pipetted through a fine pipette. All procedures were performed on a plate warmed to 38 °C. Denuded oocytes were incubated at 38 °C in RD medium supplemented with 10% FCS, 2 mM glutamine, 223 µM sodium pyruvate and 100 µM MEM non-essential amino acids (modified RD, mRD) before use. 2.3. Nuclear transfer and embryo transfer Nuclear transfers were performed as previously published (Li et al., 2006). Briefly, the metaphase plate was visualized with an inverted microscope equipped with a Spindleview system (Cambridge Research & Instrumentation Inc. Wobum, MA, USA) and was removed by a fine enucleation needle (18–20 µm outer diameter). Small cells with a smooth surface were preferentially selected and inserted into the perivitelline space of enucleated oocytes. The reconstructed couplets were electrofused with three pulses (20 µs DC 3.0 KV cm− 1) and incubated in mRD for 80 min at 38 °C. Eighty min later, nt-embryos were activated by a second set of electropulses, and incubated at 38 °C in mRD containing 5 μg/mL cycloheximide and 2 mM 6-dimethylaminopurine (Sigma chemical Co., Louis, MO, USA) for 1 h. After activation, nt-embryos were incubated in mRD for 2 h, and then transferred to 50 µl of upgraded B2 medium (Laboratoire C.C.D, Paris, France) supplemented with 2.5% FCS. The nt-embryos were either cultured to the blastocyst stage or transferred to oviducts for further development on the 2nd day. The embryo transfer receipt rabbits were injected with 80 IU hCG to induce ovulation. The time of hCG injection was 24–26 h later than was used for oocyte donor rabbits. Nt-embryos between 4–8 cells were transferred into oviducts through the infundibulum.
2.4. Caesarean and care for cloned rabbits Pregnancy was detected at 14 days by palpation. Thirtyone days after embryo transfer, caesarean deliveries were performed and newborn rabbits were maintained at 34 °C for 15 days. The cloned rabbits sucked milk from other lactating beginning 1 day after birth. 2.5. Microsatellite marker analysis Cloned rabbits were genotyped using nuclear donor fibroblasts and surrogate mothers as controls. Six primer sets were used, including 5L1C3 (AF421908), ELAMB (M91055), ASICG (AY284844), 12L1E11 (AF421941), 6L1F10 (AF421916) and 19L1A4 (AF421948) (van Haeringen et al., 1996; Korstanje et al., 2003). 3. Results In the first set of experiments, a total of 118 reconstructed couplets were produced using fetal fibroblasts and 72.9% (86/ 118) of them had fused at 30 min after electrofusion. Pronuclearlike structures appeared at 2 h after activation and 59/86 ntembryos (68.6%) entered the 2-cell stage 9–14 h later, and 21/ 86 embryos (24.4%) entered the blastocyst stage by around 72 h. In the next set of experiments, a total of 618 couplets were constructed. As shown in Table 1, a total of 289 nt-embryos at the 4–8 cell stage were transferred into oviducts of 11 pseudopregnant recipients. Six of the recipients (54.5%) were pregnant as determined by palpation 14 days after embryo transfer, and 3 of these (27.3%) carried the pregnancy to term. Caesarean delivery was performed 31 days after embryo transfer. Of three newborn rabbits, two were alive at birth. Although one died 1 h after birth, the other one lived and appeared healthy for at least 4 months (Fig. 1). Microsatellite DNA analysis confirmed that all three cloned rabbits were genetically identical to the nuclear donor fetal cells (Fig. 2). 4. Discussion It was shown previously that rabbits can be cloned from adult somatic cells through SCNT (Chesne et al., 2002; Li et al., 2006). Here we showed that fibroblasts from the rabbit fetus
Fig. 1. Rabbit cloned from a fetal fibroblast (left, 4 months old) with surrogate mother at right.
S. Li et al. / Livestock Science 122 (2009) 77–80
79
Fig. 2. Microsatellite analyses. 1–3, surrogate rabbits; 4–6, cloned rabbits; 7, donor cells. 5L1C3, 6L1F10, 12L1E11, 19L1A4, ASICG and ELAMB are six PCR primer sets used to detect DNA polymorphisms in rabbits and cells. The ruler and numbers under peaks indicate the sizes of base pairs.
also have the potential to give rise to adults. Although fetal fibroblasts can give rise to live adult rabbits, the developmental rate was lower than what has been reported for adult fibroblasts (Li et al., 2006). Using the similar protocols, 24.3% and 1.0% of fetal cell-derived embryos developed to the blastocyst stage and to term, respectively, while 45% and 3.0% of adult fibroblast derived embryos developed to the same stages, respectively (Li et al., 2006). This observation is, in fact, consistent with a previous report (Chesne et al., 2006). Nt-embryos derived from fetal fibroblasts developed poorly during post-implantation development, resulting in a lower birth rate than among embryos obtained by other authors from cumulus cells (Chesne et al., 2002). Only one rabbit was born from 42 recipient mothers transferred with 765 nt-embryos (0.13%, as compared to 1.5% for cumulus cells), and this rabbit died 1 day after birth. Nonetheless, our efforts are the first successful attempts reported using fetal fibroblast donors. A few technical improvements may have contributed to the success. First, micromanipulation under the “Spindle View” avoided damage caused by exposure to Hoechst dye and ultraviolet. Second, since the metaphase plate of the oocyte disappears at temperatures below 28 °C for 20 min, we performed all ex vivo manipulations at 38 °C to avoid pre-activation of the oocytes. Finally, we superovulated rabbits with PMSG, instead of FSH for 3 days and collected oocytes 14 h after hCG injection. Acknowledgements The study was supported by grants from the National Natural Science Foundation of China (No: 30040003), the
National Transgenic Foundation of China (2007AA10Z1A1), and the China Postdoctoral Science Foundation (20060400541). References Carney, E.W., Foote, R.H., 1991. Improved development of rabbit one-cell embryos to the hatching blastocyst stage by culture in a defined, proteinfree culture medium. J. Reprod. Fertil. 91, 113–123. Chesne, P., Adenot, P.G., Viglietta, C., Baratte, M., Boulanger, L., Renard, J.P., 2002. Cloned rabbits produced by nuclear transfer from adult somatic cells. Nat. Biotechnol. 20, 366–369. Chesne, P., Chrenek, P., Challa-Jacques, M., Daniel, N., Boulanger, L., Vignon, X., Adenot, P.G., Renard, J.-P., 2006. Cloning in the rabbit: present situation and prospects. In: Inui, A. (Ed.), Epigenetic Risk of Cloning. CRC, pp. 71–96. Cibelli, J.B., Stice, S.L., Golueke, P.J., Kane, J.J., Jerry, J., Blackwell, C., Ponce de Leon, F.A., Robl, J.M., 1998. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280, 1256–1258. Dinnyes, A., Dai, Y., Barber, M., Liu, L., Xu, J., Zhou, P., Yang, X., 2001. Development of cloned embryos from adult rabbit fibroblasts: effect of activation treatment and donor cell preparation. Biol. Reprod. 64, 257–263. Inoue, K., Ogonuki, N., Yamamoto, Y., Noguchi, Y., Takeiri, S., Nakata, K., Miki, H., Kurome, M., Nagashima, H., Ogura, A., 2002. Improved postimplantation development of rabbit nuclear transfer embryos by activation with inositol 1,4,5-trisphosphate. Cloning Stem Cells 4, 311–317. Korstanje, R., Gillissen, G.F., Versteeg, S.A., van Oost, B.A., Bosma, A.A., RogelGaillard, C., van Zutphen, L.F., van Lith, H.A., 2003. Mapping of rabbit microsatellite markers using chromosome-specific libraries. J. Hered. 94, 161–169. Lai, L., Kolber-Simonds, D., Park, K.W., Cheong, H.T., Greenstein, J.L., Im, G.S., Samuel, M., Bonk, A., Rieke, A., Day, B.N., Murphy, C.N., Carter, D.B., Hawley, R.J., Prather, R.S., 2002. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 295, 1089–1092. Li, S., Chen, X., Fang, Z., Shi, J., Sheng, H.Z., 2006. Rabbits generated from fibroblasts through nuclear transfer. Reproduction 131, 1085–1090. Mitalipov, S.M., White, K.L., Farrar, V.R., Morrey, J., Reed, W.A., 1999. Development of nuclear transfer and parthenogenetic rabbit embryos activated with inositol 1,4,5-trisphosphate. Biol. Reprod. 60, 821–827.
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S. Li et al. / Livestock Science 122 (2009) 77–80
Ogura, A., Inoue, K., Ogonuki, N., Noguchi, A., Takano, K., Nagano, R., Suzuki, O., Lee, J., Ishino, F., Matsuda, J., 2000. Production of male cloned mice from fresh, cultured, and cryopreserved immature Sertoli cells. Biol. Reprod. 62, 1579–1584. Ramsoondar, J.J., Machaty, Z., Costa, C., Williams, B.L., Fodor, W.L., Bondioli, K.R., 2003. Production of alpha 1,3-galactosyltransferase-knockout cloned pigs expressing human alpha 1,2-fucosylosyltransferase. Biol. Reprod. 69, 437–445. Schnieke, A.E., Kind, A.J., Ritchie, W.A., Mycock, K., Scott, A.R., Ritchie, M., Wilmut, I., Colman, A., Campbell, K.H., 1997. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 278, 2130–2133.
van Haeringen, W.A., den Bieman, M., van Zutphen, L.F., van Lith, H.A., 1996. Polymorphic microsatellite DNA markers in the rabbit (Oryctolagus cuniculus). J. Exp. Anim. Sci. 38, 49–57. Yang, F., Hao, R., Kessler, B., Brem, G., Wolf, E., Zakhartchenko, V., 2007. Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation. Reproduction 133, 219–230. Yin, X.J., Kato, Y., Tsunoda, Y., 2002. Effect of enucleation procedures and maturation conditions on the development of nuclear-transferred rabbit oocytes receiving male fibroblast cells. Reproduction 124, 41–47.
Contents lists available at ScienceDirect
Livestock Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i v s c i
Short communication
Cloning rabbits from fetal fibroblasts Shangang Li a,1, Xuejin Chen b,1, Jianjun Shi b, Yi Guo b, Chunguang Yin a, Lixin Du a, Hui Z. Sheng b,⁎ a b
National Center for Molecular Genetics and Breeding of Animal, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, PR China Center for Developmental Biology, Xinhua Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, PR China
a r t i c l e
i n f o
Article history: Received 12 May 2008 Received in revised form 3 July 2008 Accepted 4 July 2008 Keywords: Rabbit Nuclear transfer Fetal fibroblast Reproduction
a b s t r a c t Live rabbits were generated through nuclear transfer using adult cells as nuclear donors. We showed that, in addition to adult cell nuclei, cultured fetal rabbit fibroblasts could support fullterm embryonic development following nuclear transfer to enucleated oocytes. Following nuclear transfer, 24.4% (21/86) of resulting embryos developed to the blastocyst stage, and 289 embryos were transferred to oviducts of 11 recipient mothers, resulting in 6 pregnancies. Three mothers carried the pregnancy to term, and three newborn rabbits were subsequently delivered by caesarian, one of which survived for more than 4 months. DNA analyses confirmed that all 3 rabbits were genetically identical to fetal donor cells. This study demonstrated that rabbits could be cloned from fetal fibroblasts, although at a lower frequency than when using adult somatic cells as nuclear donors. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Somatic cell nuclear transfer (SCNT) offers new opportunities for species production and preservation. The rabbit is the preferred model organism for many biomedical and pharmaceutical studies. Improving the efficiency of rabbit cloning and understanding mechanisms underlying effective cloning are important goals in the field (Mitalipov et al., 1999; Ogura et al., 2000; Dinnyes et al., 2001; Inoue et al., 2002; Yin et al., 2002; Yang et al., 2007). Rabbits have been cloned from nuclei of freshly isolated cumulus cells (Chesne et al., 2002) and adult fibroblasts (Li et al., 2006). Another goal of cloning is to develop efficient methods for introducing genes into rabbits for a variety of applications. In other species, fetal cells are often used for transgenic manipulation (Schnieke et al.,1997; Cibelli et al., 1998; Lai et al., 2002; Ramsoondar et al., 2003). Thus, methods for cloning rabbits from fetal cells would contribute to advancements in rabbit transgenesis as well. However, similar experiments in the rabbit have not yet been reported (Chesne et al., 2006).
⁎ Corresponding author. Center for Developmental Biology, Xinhua Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, 200092, PR China. Tel./fax: +86 21 55570017. E-mail address: [email protected] (H.Z. Sheng). 1 These authors contributed equally to the paper. 1871-1413/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2008.07.019
Here, we present methods for cloning live rabbits from fetal fibroblasts. This will contribute to extending the use of rabbit models by transgenesis. 2. Materials and methods Culture media were purchased from Gibco Invitrogen (Grand Island, NY, USA), unless otherwise noted. Protocols for animal care, SCNT, embryo transfer and Caesarean delivery of rabbits were approved by the Ethics Committee, Xinhua Hospital, Shanghai Jiao Tong University, School of Medicine. 2.1. Preparation of fetal fibroblasts Skin was isolated from the back of a New Zealand White fetus on embryonic day 20 (E20). The tissue was cut into 1 mm cubes, digested with 0.05% trypsin/EDTA for 20 min at 37 °C, transferred into a 25-cm2 culture flask containing 6–8 ml DMEM supplemented with 10% fetal calf serum (FCS, Hyclone Co., Salt Lake City, UT, USA) and cultured at 37 °C in 5% CO2. After approximately 6 days, cells grew out from explants and covered the flask surface. Cells were then treated with 0.05% trypsin/ EDTA and passaged to 3 new flasks (passage 1). The fetal fibroblasts were frozen in liquid nitrogen at passage 3 for future use. To prepare donor cells, 105 fibroblasts from 5–15 passages
78
S. Li et al. / Livestock Science 122 (2009) 77–80
Table 1 A summary in development of nuclear transfer embryos in vivo Sample size
Developmental rate
No. reconstructed No. fused couplets (% from reconstructed) No. cleaved embryos (% from fused) No. transferred embryos No. recipient mothers No. recipients pregnant (% of total recipients) No. recipients delivering (% of total recipients) No. full-term fetuses (% from transferred embryos) No. living fetuses (% from transferred embryos) No. alive at weaning (% from transferred embryos)
618 456 (73.8) 335 (73.5) 289 11 6 (54.5) 3 (27.3) 3 (1.0) 2 (0.7) 1 (0.3)
were cultured to confluence in a 3.5 cm dish and then were in DMEM supplemented with 0.5% FCS for 2–4 days. Cells were harvested with trypsin, resuspended in DMEM with 10% FCS, kept at 4 °C to prevent aggregation. Before nuclear transfer, donor cells were incubated at 37 °C for 10 min, and transferred into micro-drops for microinjection. 2.2. Oocyte collection Adult female White New Zealand rabbits were superovulated as previously published (Li et al., 2006). Oviducts were collected 14 h after hCG injection, flushed with prewarmed RD medium (Carney and Foote, 1991) supplemented with 10% (v/v) FCS and 10 mM Hepes (Hepes balanced RD, hRD). Cumulus cells were removed by exposure to 100 IU/ml hyaluronidase (Sigma chemical Co.) in hRD medium for 10 min and then pipetted through a fine pipette. All procedures were performed on a plate warmed to 38 °C. Denuded oocytes were incubated at 38 °C in RD medium supplemented with 10% FCS, 2 mM glutamine, 223 µM sodium pyruvate and 100 µM MEM non-essential amino acids (modified RD, mRD) before use. 2.3. Nuclear transfer and embryo transfer Nuclear transfers were performed as previously published (Li et al., 2006). Briefly, the metaphase plate was visualized with an inverted microscope equipped with a Spindleview system (Cambridge Research & Instrumentation Inc. Wobum, MA, USA) and was removed by a fine enucleation needle (18–20 µm outer diameter). Small cells with a smooth surface were preferentially selected and inserted into the perivitelline space of enucleated oocytes. The reconstructed couplets were electrofused with three pulses (20 µs DC 3.0 KV cm− 1) and incubated in mRD for 80 min at 38 °C. Eighty min later, nt-embryos were activated by a second set of electropulses, and incubated at 38 °C in mRD containing 5 μg/mL cycloheximide and 2 mM 6-dimethylaminopurine (Sigma chemical Co., Louis, MO, USA) for 1 h. After activation, nt-embryos were incubated in mRD for 2 h, and then transferred to 50 µl of upgraded B2 medium (Laboratoire C.C.D, Paris, France) supplemented with 2.5% FCS. The nt-embryos were either cultured to the blastocyst stage or transferred to oviducts for further development on the 2nd day. The embryo transfer receipt rabbits were injected with 80 IU hCG to induce ovulation. The time of hCG injection was 24–26 h later than was used for oocyte donor rabbits. Nt-embryos between 4–8 cells were transferred into oviducts through the infundibulum.
2.4. Caesarean and care for cloned rabbits Pregnancy was detected at 14 days by palpation. Thirtyone days after embryo transfer, caesarean deliveries were performed and newborn rabbits were maintained at 34 °C for 15 days. The cloned rabbits sucked milk from other lactating beginning 1 day after birth. 2.5. Microsatellite marker analysis Cloned rabbits were genotyped using nuclear donor fibroblasts and surrogate mothers as controls. Six primer sets were used, including 5L1C3 (AF421908), ELAMB (M91055), ASICG (AY284844), 12L1E11 (AF421941), 6L1F10 (AF421916) and 19L1A4 (AF421948) (van Haeringen et al., 1996; Korstanje et al., 2003). 3. Results In the first set of experiments, a total of 118 reconstructed couplets were produced using fetal fibroblasts and 72.9% (86/ 118) of them had fused at 30 min after electrofusion. Pronuclearlike structures appeared at 2 h after activation and 59/86 ntembryos (68.6%) entered the 2-cell stage 9–14 h later, and 21/ 86 embryos (24.4%) entered the blastocyst stage by around 72 h. In the next set of experiments, a total of 618 couplets were constructed. As shown in Table 1, a total of 289 nt-embryos at the 4–8 cell stage were transferred into oviducts of 11 pseudopregnant recipients. Six of the recipients (54.5%) were pregnant as determined by palpation 14 days after embryo transfer, and 3 of these (27.3%) carried the pregnancy to term. Caesarean delivery was performed 31 days after embryo transfer. Of three newborn rabbits, two were alive at birth. Although one died 1 h after birth, the other one lived and appeared healthy for at least 4 months (Fig. 1). Microsatellite DNA analysis confirmed that all three cloned rabbits were genetically identical to the nuclear donor fetal cells (Fig. 2). 4. Discussion It was shown previously that rabbits can be cloned from adult somatic cells through SCNT (Chesne et al., 2002; Li et al., 2006). Here we showed that fibroblasts from the rabbit fetus
Fig. 1. Rabbit cloned from a fetal fibroblast (left, 4 months old) with surrogate mother at right.
S. Li et al. / Livestock Science 122 (2009) 77–80
79
Fig. 2. Microsatellite analyses. 1–3, surrogate rabbits; 4–6, cloned rabbits; 7, donor cells. 5L1C3, 6L1F10, 12L1E11, 19L1A4, ASICG and ELAMB are six PCR primer sets used to detect DNA polymorphisms in rabbits and cells. The ruler and numbers under peaks indicate the sizes of base pairs.
also have the potential to give rise to adults. Although fetal fibroblasts can give rise to live adult rabbits, the developmental rate was lower than what has been reported for adult fibroblasts (Li et al., 2006). Using the similar protocols, 24.3% and 1.0% of fetal cell-derived embryos developed to the blastocyst stage and to term, respectively, while 45% and 3.0% of adult fibroblast derived embryos developed to the same stages, respectively (Li et al., 2006). This observation is, in fact, consistent with a previous report (Chesne et al., 2006). Nt-embryos derived from fetal fibroblasts developed poorly during post-implantation development, resulting in a lower birth rate than among embryos obtained by other authors from cumulus cells (Chesne et al., 2002). Only one rabbit was born from 42 recipient mothers transferred with 765 nt-embryos (0.13%, as compared to 1.5% for cumulus cells), and this rabbit died 1 day after birth. Nonetheless, our efforts are the first successful attempts reported using fetal fibroblast donors. A few technical improvements may have contributed to the success. First, micromanipulation under the “Spindle View” avoided damage caused by exposure to Hoechst dye and ultraviolet. Second, since the metaphase plate of the oocyte disappears at temperatures below 28 °C for 20 min, we performed all ex vivo manipulations at 38 °C to avoid pre-activation of the oocytes. Finally, we superovulated rabbits with PMSG, instead of FSH for 3 days and collected oocytes 14 h after hCG injection. Acknowledgements The study was supported by grants from the National Natural Science Foundation of China (No: 30040003), the
National Transgenic Foundation of China (2007AA10Z1A1), and the China Postdoctoral Science Foundation (20060400541). References Carney, E.W., Foote, R.H., 1991. Improved development of rabbit one-cell embryos to the hatching blastocyst stage by culture in a defined, proteinfree culture medium. J. Reprod. Fertil. 91, 113–123. Chesne, P., Adenot, P.G., Viglietta, C., Baratte, M., Boulanger, L., Renard, J.P., 2002. Cloned rabbits produced by nuclear transfer from adult somatic cells. Nat. Biotechnol. 20, 366–369. Chesne, P., Chrenek, P., Challa-Jacques, M., Daniel, N., Boulanger, L., Vignon, X., Adenot, P.G., Renard, J.-P., 2006. Cloning in the rabbit: present situation and prospects. In: Inui, A. (Ed.), Epigenetic Risk of Cloning. CRC, pp. 71–96. Cibelli, J.B., Stice, S.L., Golueke, P.J., Kane, J.J., Jerry, J., Blackwell, C., Ponce de Leon, F.A., Robl, J.M., 1998. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280, 1256–1258. Dinnyes, A., Dai, Y., Barber, M., Liu, L., Xu, J., Zhou, P., Yang, X., 2001. Development of cloned embryos from adult rabbit fibroblasts: effect of activation treatment and donor cell preparation. Biol. Reprod. 64, 257–263. Inoue, K., Ogonuki, N., Yamamoto, Y., Noguchi, Y., Takeiri, S., Nakata, K., Miki, H., Kurome, M., Nagashima, H., Ogura, A., 2002. Improved postimplantation development of rabbit nuclear transfer embryos by activation with inositol 1,4,5-trisphosphate. Cloning Stem Cells 4, 311–317. Korstanje, R., Gillissen, G.F., Versteeg, S.A., van Oost, B.A., Bosma, A.A., RogelGaillard, C., van Zutphen, L.F., van Lith, H.A., 2003. Mapping of rabbit microsatellite markers using chromosome-specific libraries. J. Hered. 94, 161–169. Lai, L., Kolber-Simonds, D., Park, K.W., Cheong, H.T., Greenstein, J.L., Im, G.S., Samuel, M., Bonk, A., Rieke, A., Day, B.N., Murphy, C.N., Carter, D.B., Hawley, R.J., Prather, R.S., 2002. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 295, 1089–1092. Li, S., Chen, X., Fang, Z., Shi, J., Sheng, H.Z., 2006. Rabbits generated from fibroblasts through nuclear transfer. Reproduction 131, 1085–1090. Mitalipov, S.M., White, K.L., Farrar, V.R., Morrey, J., Reed, W.A., 1999. Development of nuclear transfer and parthenogenetic rabbit embryos activated with inositol 1,4,5-trisphosphate. Biol. Reprod. 60, 821–827.
80
S. Li et al. / Livestock Science 122 (2009) 77–80
Ogura, A., Inoue, K., Ogonuki, N., Noguchi, A., Takano, K., Nagano, R., Suzuki, O., Lee, J., Ishino, F., Matsuda, J., 2000. Production of male cloned mice from fresh, cultured, and cryopreserved immature Sertoli cells. Biol. Reprod. 62, 1579–1584. Ramsoondar, J.J., Machaty, Z., Costa, C., Williams, B.L., Fodor, W.L., Bondioli, K.R., 2003. Production of alpha 1,3-galactosyltransferase-knockout cloned pigs expressing human alpha 1,2-fucosylosyltransferase. Biol. Reprod. 69, 437–445. Schnieke, A.E., Kind, A.J., Ritchie, W.A., Mycock, K., Scott, A.R., Ritchie, M., Wilmut, I., Colman, A., Campbell, K.H., 1997. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 278, 2130–2133.
van Haeringen, W.A., den Bieman, M., van Zutphen, L.F., van Lith, H.A., 1996. Polymorphic microsatellite DNA markers in the rabbit (Oryctolagus cuniculus). J. Exp. Anim. Sci. 38, 49–57. Yang, F., Hao, R., Kessler, B., Brem, G., Wolf, E., Zakhartchenko, V., 2007. Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation. Reproduction 133, 219–230. Yin, X.J., Kato, Y., Tsunoda, Y., 2002. Effect of enucleation procedures and maturation conditions on the development of nuclear-transferred rabbit oocytes receiving male fibroblast cells. Reproduction 124, 41–47.