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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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Construction of a rabbit Oct4 promoter-based EGFP vector.(A) Diagram of the pROP2-EGFP vector containing a 3.0 kb rabbit Oct4 promoter followed by EGFP in our experiments. (B) Partial EGFP expression in Rb-ESCs after transfection with pROP2-EGFP. (C) EGFP images after the transfection of CMV-GFP, PGK-EGFP, pROP2-EGFP and empty vector into murine ES cell line (R1) and rabbit fibroblasts (RFFs). Scale bars = 50 µm.
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pone-0109728-g001: Construction of a rabbit Oct4 promoter-based EGFP vector.(A) Diagram of the pROP2-EGFP vector containing a 3.0 kb rabbit Oct4 promoter followed by EGFP in our experiments. (B) Partial EGFP expression in Rb-ESCs after transfection with pROP2-EGFP. (C) EGFP images after the transfection of CMV-GFP, PGK-EGFP, pROP2-EGFP and empty vector into murine ES cell line (R1) and rabbit fibroblasts (RFFs). Scale bars = 50 µm.

Mentions: The sequences of the rabbit Oct4 gene and its promoter have been previously reported [20]. The rabbit Oct4 promoter contains four conserved regions (CR1, CR2, CR3 and CR4) and two enhancers. We selected a 3.0 kb-long sequence that contains the four conserved regions, the distal enhancer (DE), the proximal enhancer (PE) and the first exon as the promoter (Fig. 1A). The promoter was amplified from a New Zealand white rabbit by genomic PCR and then cloned into the upstream of the EGFP-coding region to generate the pROP2-EGFP vector.


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)

Construction of a rabbit Oct4 promoter-based EGFP vector.(A) Diagram of the pROP2-EGFP vector containing a 3.0 kb rabbit Oct4 promoter followed by EGFP in our experiments. (B) Partial EGFP expression in Rb-ESCs after transfection with pROP2-EGFP. (C) EGFP images after the transfection of CMV-GFP, PGK-EGFP, pROP2-EGFP and empty vector into murine ES cell line (R1) and rabbit fibroblasts (RFFs). Scale bars = 50 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0109728-g001: Construction of a rabbit Oct4 promoter-based EGFP vector.(A) Diagram of the pROP2-EGFP vector containing a 3.0 kb rabbit Oct4 promoter followed by EGFP in our experiments. (B) Partial EGFP expression in Rb-ESCs after transfection with pROP2-EGFP. (C) EGFP images after the transfection of CMV-GFP, PGK-EGFP, pROP2-EGFP and empty vector into murine ES cell line (R1) and rabbit fibroblasts (RFFs). Scale bars = 50 µm.
Mentions: The sequences of the rabbit Oct4 gene and its promoter have been previously reported [20]. The rabbit Oct4 promoter contains four conserved regions (CR1, CR2, CR3 and CR4) and two enhancers. We selected a 3.0 kb-long sequence that contains the four conserved regions, the distal enhancer (DE), the proximal enhancer (PE) and the first exon as the promoter (Fig. 1A). The promoter was amplified from a New Zealand white rabbit by genomic PCR and then cloned into the upstream of the EGFP-coding region to generate the pROP2-EGFP vector.

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