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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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Analysis of NT fetuses.(A) Two cloned fetuses (named 1## and 2##) derived from 17# transgenic clones by SCNT at 15 days after transplantation. (B) Genomic PCR analysis of fetuses 1## and 2## stably transfected with pROP2-EGFP. NC: negative control. (C) EGFP expressed in genital ridges isolated from 2## fetus but not in other tissues, such as intestinal tissues. Scale bars = 1 mm. (D) EGFP reactivated in morulae and blastocysts after the second SCNT was performed using Oct4-EGFP transgenic fibroblasts isolated from fetus 1## and 2## as donors. Parthenogenetic blastocysts did not express EGFP. Scale bars = 100 µm.
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pone-0109728-g003: Analysis of NT fetuses.(A) Two cloned fetuses (named 1## and 2##) derived from 17# transgenic clones by SCNT at 15 days after transplantation. (B) Genomic PCR analysis of fetuses 1## and 2## stably transfected with pROP2-EGFP. NC: negative control. (C) EGFP expressed in genital ridges isolated from 2## fetus but not in other tissues, such as intestinal tissues. Scale bars = 1 mm. (D) EGFP reactivated in morulae and blastocysts after the second SCNT was performed using Oct4-EGFP transgenic fibroblasts isolated from fetus 1## and 2## as donors. Parthenogenetic blastocysts did not express EGFP. Scale bars = 100 µm.

Mentions: Forty-eight 17# nuclear-transferred embryos were transferred into three surrogate rabbits at 1 day after activation. At 15 days after embryo transfer, two fetuses (marked 1## and 2##) were retrieved from two surrogates by caesarean section (Fig. 3A). Genomic PCR analysis confirmed that both of these fetuses were integrated with the exogenous pROP2-EGFP vector (Fig. 3B). Genital ridges were collected and observed under a microscope. EGFP expression was found in the genital ridges of both fetuses, but not in other tissues, such as intestinal tissues (Fig. 3C). Neural stem cells (Rb-NSCs) and RFFs were isolated from the fetuses of the two transgenic rabbits. Fibroblasts were then used as donor cells to perform a second round of nuclear transfer. EGFP expression was also found in the morula and ICM of blastocysts (Fig. 3D). This result was also observed in the response of the embryos derived from the original transgenic fibroblasts. Therefore, the Oct4 promoter was reactivated.


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)

Analysis of NT fetuses.(A) Two cloned fetuses (named 1## and 2##) derived from 17# transgenic clones by SCNT at 15 days after transplantation. (B) Genomic PCR analysis of fetuses 1## and 2## stably transfected with pROP2-EGFP. NC: negative control. (C) EGFP expressed in genital ridges isolated from 2## fetus but not in other tissues, such as intestinal tissues. Scale bars = 1 mm. (D) EGFP reactivated in morulae and blastocysts after the second SCNT was performed using Oct4-EGFP transgenic fibroblasts isolated from fetus 1## and 2## as donors. Parthenogenetic blastocysts did not express EGFP. Scale bars = 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0109728-g003: Analysis of NT fetuses.(A) Two cloned fetuses (named 1## and 2##) derived from 17# transgenic clones by SCNT at 15 days after transplantation. (B) Genomic PCR analysis of fetuses 1## and 2## stably transfected with pROP2-EGFP. NC: negative control. (C) EGFP expressed in genital ridges isolated from 2## fetus but not in other tissues, such as intestinal tissues. Scale bars = 1 mm. (D) EGFP reactivated in morulae and blastocysts after the second SCNT was performed using Oct4-EGFP transgenic fibroblasts isolated from fetus 1## and 2## as donors. Parthenogenetic blastocysts did not express EGFP. Scale bars = 100 µm.
Mentions: Forty-eight 17# nuclear-transferred embryos were transferred into three surrogate rabbits at 1 day after activation. At 15 days after embryo transfer, two fetuses (marked 1## and 2##) were retrieved from two surrogates by caesarean section (Fig. 3A). Genomic PCR analysis confirmed that both of these fetuses were integrated with the exogenous pROP2-EGFP vector (Fig. 3B). Genital ridges were collected and observed under a microscope. EGFP expression was found in the genital ridges of both fetuses, but not in other tissues, such as intestinal tissues (Fig. 3C). Neural stem cells (Rb-NSCs) and RFFs were isolated from the fetuses of the two transgenic rabbits. Fibroblasts were then used as donor cells to perform a second round of nuclear transfer. EGFP expression was also found in the morula and ICM of blastocysts (Fig. 3D). This result was also observed in the response of the embryos derived from the original transgenic fibroblasts. Therefore, the Oct4 promoter was reactivated.

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