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Establishment of a rabbit Oct4 promoter-based EGFP reporter system.

Quan L, Chen Y, Song J, Yan Q, Zhang Q, Lai S, Fan N, Xin J, Zou Q, Lai L - PLoS ONE (2014)

Bottom Line: The EGFP expression was detected in the blastocysts and genital ridges of SCNT fetuses.Fibroblasts and neural stem cells (NSCs) were derived from the SCNT fetuses.EGFP was also reactivated in blastocysts after the second SCNT, and induced pluripotent stem cells (iPSCs) were obtained after reprogramming using Yamanaka's factors.

View Article: PubMed Central - PubMed

Affiliation: Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Science, Jilin University, Changchun, China.

ABSTRACT
Rabbits are commonly used as laboratory animal models to investigate human diseases and phylogenetic development. However, pluripotent stem cells that contribute to germline transmission have yet to be established in rabbits. The transcription factor Oct4, also known as Pou5f1, is considered essential for the maintenance of the pluripotency of stem cells. Hence, pluripotent cells can be identified by monitoring Oct4 expression using a well-established Oct4 promoter-based reporter system. This study developed a rabbit Oct4 promoter-based enhanced green fluorescent protein (EGFP) reporter system by transfecting pROP2-EGFP into rabbit fetal fibroblasts (RFFs). The transgenic RFFs were used as donor cells for somatic cell nuclear transfer (SCNT). The EGFP expression was detected in the blastocysts and genital ridges of SCNT fetuses. Fibroblasts and neural stem cells (NSCs) were derived from the SCNT fetuses. EGFP was also reactivated in blastocysts after the second SCNT, and induced pluripotent stem cells (iPSCs) were obtained after reprogramming using Yamanaka's factors. The results above indicated that a rabbit reporter system used to monitor the differentiating status of cells was successfully developed.

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Selection of RFF donor cells stably transfected with pROP2-EGFP for nuclear transfer.(A) PCR analysis of rabbit fibroblasts stably transfected with pROP2-EGFP. (+: positive control; –: negative control; 1–19: G418-resistant clones; 3#, 4#, 5#, 11#, 12#, 13# and 17#: positive transgenic clones). (B) EGFP expressed in NT embryos using 13#, 17# fibroblasts as nuclear donors, while parthenogenetic blastocysts did not express EGFP. The arrow points ICM. PA: parthenogenetic embryos. (C) Immunoflorescent assay showed that EGFP co-expressed with Oct4 in the ICM from NT embryos at the late blastocyst stage. Scale bars = 100 µm.
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pone-0109728-g002: Selection of RFF donor cells stably transfected with pROP2-EGFP for nuclear transfer.(A) PCR analysis of rabbit fibroblasts stably transfected with pROP2-EGFP. (+: positive control; –: negative control; 1–19: G418-resistant clones; 3#, 4#, 5#, 11#, 12#, 13# and 17#: positive transgenic clones). (B) EGFP expressed in NT embryos using 13#, 17# fibroblasts as nuclear donors, while parthenogenetic blastocysts did not express EGFP. The arrow points ICM. PA: parthenogenetic embryos. (C) Immunoflorescent assay showed that EGFP co-expressed with Oct4 in the ICM from NT embryos at the late blastocyst stage. Scale bars = 100 µm.

Mentions: RFFs were transfected with the pROP2-EGFP vector by electroporation and selected using G418 for 2 weeks. A total of 135 cell clones were harvested for passage, and 19 cell lines were established. Seven cell lines (marked 3#, 4#, 5#, 11#, 12#, 13# and 17#) were positive for pROP2-EGFP vector integration as confirmed by genomic PCR (Fig. 2A). We selected 17# fibroblasts as nuclear donors for SCNT. The cleavage rate of oocytes was 90.9% (40/44) at 24 hours after activation, and the blastocyst rate reached 40.9% (18/44) (Table 1), indicating that the in vitro development of nuclear-transferred embryos was not affected by the integration of the pROP2-EGFP vector. The morulae and blastocysts were examined via fluorescence microscopy. EGFP expression was found in the morulae and blastocysts (8/18, 44.4%) but not in the parthenogenetic (PA) counterpart embryos. Other transgenic fibroblasts, such as 13# fibroblasts, were also used for SCNT. EGFP was observed when the embryos developed to morula and ICM of blastocysts. Trophoblast cells also expressed weak EGFP fluorescence at the early blastocyst stage (Fig. 2B). Immunostaining results showed that EGFP and Oct4 were co-expressed in the ICM of the late blastocyst stage, while both were inactivated in trophoblast cells (Fig. 2C). These results indicated that the Oct4 promoter was activated when somatic cells with Oct4 promoter EGFP vector were reprogrammed into pluripotent cells by the cytoplasm of oocytes through nuclear transfer.


Establishment of a rabbit Oct4 promoter-based EGFP reporter system.

Quan L, Chen Y, Song J, Yan Q, Zhang Q, Lai S, Fan N, Xin J, Zou Q, Lai L - PLoS ONE (2014)

Selection of RFF donor cells stably transfected with pROP2-EGFP for nuclear transfer.(A) PCR analysis of rabbit fibroblasts stably transfected with pROP2-EGFP. (+: positive control; –: negative control; 1–19: G418-resistant clones; 3#, 4#, 5#, 11#, 12#, 13# and 17#: positive transgenic clones). (B) EGFP expressed in NT embryos using 13#, 17# fibroblasts as nuclear donors, while parthenogenetic blastocysts did not express EGFP. The arrow points ICM. PA: parthenogenetic embryos. (C) Immunoflorescent assay showed that EGFP co-expressed with Oct4 in the ICM from NT embryos at the late blastocyst stage. Scale bars = 100 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215976&req=5

pone-0109728-g002: Selection of RFF donor cells stably transfected with pROP2-EGFP for nuclear transfer.(A) PCR analysis of rabbit fibroblasts stably transfected with pROP2-EGFP. (+: positive control; –: negative control; 1–19: G418-resistant clones; 3#, 4#, 5#, 11#, 12#, 13# and 17#: positive transgenic clones). (B) EGFP expressed in NT embryos using 13#, 17# fibroblasts as nuclear donors, while parthenogenetic blastocysts did not express EGFP. The arrow points ICM. PA: parthenogenetic embryos. (C) Immunoflorescent assay showed that EGFP co-expressed with Oct4 in the ICM from NT embryos at the late blastocyst stage. Scale bars = 100 µm.
Mentions: RFFs were transfected with the pROP2-EGFP vector by electroporation and selected using G418 for 2 weeks. A total of 135 cell clones were harvested for passage, and 19 cell lines were established. Seven cell lines (marked 3#, 4#, 5#, 11#, 12#, 13# and 17#) were positive for pROP2-EGFP vector integration as confirmed by genomic PCR (Fig. 2A). We selected 17# fibroblasts as nuclear donors for SCNT. The cleavage rate of oocytes was 90.9% (40/44) at 24 hours after activation, and the blastocyst rate reached 40.9% (18/44) (Table 1), indicating that the in vitro development of nuclear-transferred embryos was not affected by the integration of the pROP2-EGFP vector. The morulae and blastocysts were examined via fluorescence microscopy. EGFP expression was found in the morulae and blastocysts (8/18, 44.4%) but not in the parthenogenetic (PA) counterpart embryos. Other transgenic fibroblasts, such as 13# fibroblasts, were also used for SCNT. EGFP was observed when the embryos developed to morula and ICM of blastocysts. Trophoblast cells also expressed weak EGFP fluorescence at the early blastocyst stage (Fig. 2B). Immunostaining results showed that EGFP and Oct4 were co-expressed in the ICM of the late blastocyst stage, while both were inactivated in trophoblast cells (Fig. 2C). These results indicated that the Oct4 promoter was activated when somatic cells with Oct4 promoter EGFP vector were reprogrammed into pluripotent cells by the cytoplasm of oocytes through nuclear transfer.

Bottom Line: The EGFP expression was detected in the blastocysts and genital ridges of SCNT fetuses.Fibroblasts and neural stem cells (NSCs) were derived from the SCNT fetuses.EGFP was also reactivated in blastocysts after the second SCNT, and induced pluripotent stem cells (iPSCs) were obtained after reprogramming using Yamanaka's factors.

View Article: PubMed Central - PubMed

Affiliation: Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Science, Jilin University, Changchun, China.

ABSTRACT
Rabbits are commonly used as laboratory animal models to investigate human diseases and phylogenetic development. However, pluripotent stem cells that contribute to germline transmission have yet to be established in rabbits. The transcription factor Oct4, also known as Pou5f1, is considered essential for the maintenance of the pluripotency of stem cells. Hence, pluripotent cells can be identified by monitoring Oct4 expression using a well-established Oct4 promoter-based reporter system. This study developed a rabbit Oct4 promoter-based enhanced green fluorescent protein (EGFP) reporter system by transfecting pROP2-EGFP into rabbit fetal fibroblasts (RFFs). The transgenic RFFs were used as donor cells for somatic cell nuclear transfer (SCNT). The EGFP expression was detected in the blastocysts and genital ridges of SCNT fetuses. Fibroblasts and neural stem cells (NSCs) were derived from the SCNT fetuses. EGFP was also reactivated in blastocysts after the second SCNT, and induced pluripotent stem cells (iPSCs) were obtained after reprogramming using Yamanaka's factors. The results above indicated that a rabbit reporter system used to monitor the differentiating status of cells was successfully developed.

Show MeSH
Related in: MedlinePlus