Rabbits generated from fibroblasts through nuclear transfer

Li, Shangang; Chen, Xuejin; Fang, Zhenfu; Shi, Jiankui; Sheng, Hua

doi:10.1530/rep.1.01065

Cited by 44 publications

(45 citation statements)

References 28 publications

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“…Similarly, cells from tail tips of male mice performed better in NT than those of female mice (42). A 2-fold advantage in using male clones for NT has also been noted for other mammalian species (43,44). Given these collective findings, it seems likely that the difference in cloning efficiency reflects an inherent difference in female cells, which face an additional demand of reprogramming that is imposed by Xchromosome inactivation (45).…”

Section: Discussionmentioning

confidence: 78%

Mice cloned from skin cells

Li¹,

Greco

Guasch

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Adult stem cells represent unique populations of undifferentiated cells with self-renewal capacity. In many tissues, stem cells divide less often than their progeny. It has been widely speculated, but largely untested, that their undifferentiated and quiescent state may make stem cells more efficient as donors for cloning by nuclear transfer (NT). Here, we report the use of nuclei from hair follicle stem cells and other skin keratinocytes as NT donors. When keratinocyte stem cells (KSCs) were used as NT donors, 19 liveborn mice were obtained, 9 of which survived to adulthood. Embryonic keratinocytes and cumulus cells also gave rise to cloned mice. Although cloning efficiencies were similar (<6% per transferred blastocyst), success rates were consistently higher for males than for females. Adult keratinocyte stem cells were better NT donors than so-called transit amplifying (TA) keratinocytes in both sexes (1.6% vs. 0% in females and 5.4% vs. 2.8% in males). Our findings reveal skin as a source of readily accessible stem cells, the nuclei of which can be reprogrammed to the pluripotent state by exposure to the cytoplasm of unfertilized oocytes.adult stem cells ͉ embryos ͉ cloning ͉ keratinocyte ͉ nuclear transfer

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Section: Discussionmentioning

confidence: 78%

Mice cloned from skin cells

Li¹,

Greco

Guasch

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Recently, Li et al (2006) reported a higher blastocyst rate with serum-starved than with confluent rabbit fibroblasts (46 vs 21%) attributing this effect to synchronizing the cells in an inactive G0/G1 cell cycle phase. Moreover, after transfer of embryos cloned from serum-starved fibroblasts into recipients, 14 pups have been obtained and three of them survived.…”

Section: In Vitro Developmentmentioning

confidence: 99%

“…Only recently, Li et al (2006) reported production of cloned rabbits from cultured adult fibroblasts using a 'novel' SCNT protocol involving minor modifications of the previously established SCNT technique (Chesne et al 2002). However, from a total 14 born cloned rabbits 9 died shortly after birth.…”

Section: Introductionmentioning

confidence: 99%

Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation

Yang

Hao²,

Keßler³

et al. 2007

Reproduction

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The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres from in vivo fertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P!0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF, P!0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that in in vivo fertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and may be a useful epigenetic mark to predict efficiency of SCNT in rabbits.

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“…Given their frequent use in research, the creation of genetically modified rabbit lines is highly desirable. The development of somatic cell nuclear transfer (SCNT) technology in rabbits has been the focus of much recent research (Mitalipov et al 1999;Dinnyes et al 2001;Inoue et al 2002;Yin et al 2002;Yang et al 2007), and rabbits have been successfully cloned from freshly isolated cumulus cells (Chesne et al 2002) and adult male fibroblasts (Li et al 2006). However, when rabbit cells have been used as donor cells after gene transfection, the results of SCNT have been poor, and although transgenic chimeric rabbits have been produced by a variety of means (Ogura et al 2000;Skrzyszowska et al 2006), there have been no reports of the birth of cloned transgenic rabbit from SCNT.…”

Section: Introductionmentioning

confidence: 99%

Transgene expression of enhanced green fluorescent protein in cloned rabbits generated from in vitro-transfected adult fibroblasts

Li¹,

Guo

Shi

et al. 2008

Transgenic Res

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Live rabbits have previously been generated through nuclear transfer using adult cells as nuclear donors. We demonstrated in this study that transfected adult rabbit fibroblasts are also capable of supporting full-term development. The fibroblasts were transfected with a pEGFP-C1 plasmid using lipofectamine() 2000, and the transgenic cells were derived from conditioned medium. The transgenic fibroblasts were cultured until confluent and then serum-starved prior to be used as nuclear donors. After nuclear transfer and activation, 22% (12/55) of the transgenic cloned embryos developed to the blastocyst stage. A total of 114 embryos at the 4- to 8-cell stage were transferred to the oviducts of 8 pseudo-pregnant mothers; 5 of these animals became pregnant, and 3 of the 5 mother rabbits carried the pregnancy to term. Caesarean section was performed on the 3 pregnant mothers, yielding 4 kits, one of which has survived for more than 9 months. Green fluorescence could be detected in the toenails of the living cloned rabbit and the offspring from the living cloned rabbit under ultraviolet light. DNA analyses confirmed that all 4 cloned rabbits were genetically identical to the transgenic donor cells, and that they all carried the EGFP gene. The present study demonstrated that transgenic rabbits can be generated through nuclear transfer. These results may facilitate future developments in the genetic engineering of rabbits.

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Rabbits generated from fibroblasts through nuclear transfer

Cited by 44 publications

References 28 publications

Mice cloned from skin cells

Mice cloned from skin cells

Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation

Transgene expression of enhanced green fluorescent protein in cloned rabbits generated from in vitro-transfected adult fibroblasts

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