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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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Generation of Rb-iPSCs from transgenic Rb-NSCs.(A) Experimental outline for generating Rb-iPSCs from Rb-NSCs. (B) The morphology of NSCs changed to multi-layer compacted clones after 11 days of induction using human transcription factors Oct4, Klf4, Sox2 and c-Myc. (C) EGFP activated in several Rb-iPS colonies in 3i medium (hLIF plus 3i) and BL medium (hLIF plus bFGF). (D) Rb-iPSCs (Rb-iPSC bl13 cultured in BL medium and Rb-iPSC i5 cultured in 3i medium) maintained a normal karyotype at 15 passages. (E) Immunostaining assay showed the expressed Oct4 in Rb-iPSCs. (F) RT-PCR result demonstrating that endogenous Oct4 was not expressed in all the proliferated colonies and that exogenous Oct4 was continuously expressed in Rb-iPSC colonies. (G) Expressed markers of the three germ layers after Rb-iPSCs differentiated in vitro. Tuj1 (ectoderm); AFP (endoderm); and smooth muscle actin (mesoderm). Scale bars = 50 µm.
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pone-0109728-g004: Generation of Rb-iPSCs from transgenic Rb-NSCs.(A) Experimental outline for generating Rb-iPSCs from Rb-NSCs. (B) The morphology of NSCs changed to multi-layer compacted clones after 11 days of induction using human transcription factors Oct4, Klf4, Sox2 and c-Myc. (C) EGFP activated in several Rb-iPS colonies in 3i medium (hLIF plus 3i) and BL medium (hLIF plus bFGF). (D) Rb-iPSCs (Rb-iPSC bl13 cultured in BL medium and Rb-iPSC i5 cultured in 3i medium) maintained a normal karyotype at 15 passages. (E) Immunostaining assay showed the expressed Oct4 in Rb-iPSCs. (F) RT-PCR result demonstrating that endogenous Oct4 was not expressed in all the proliferated colonies and that exogenous Oct4 was continuously expressed in Rb-iPSC colonies. (G) Expressed markers of the three germ layers after Rb-iPSCs differentiated in vitro. Tuj1 (ectoderm); AFP (endoderm); and smooth muscle actin (mesoderm). Scale bars = 50 µm.

Mentions: We used Rb-NSCs isolated from the Oct4-EGFP transgenic fetuses to tentatively generate Rb-iPSCs through the infection of lentiviral vectors that encode four human transcription factors (Oct4, Sox2, Klf4 and c-Myc) (Fig. 4A). The cellular morphology changed at 4 days after infection, and iPS-like colonies were first observed after 8 days. The morphological characteristics of iPS-like colonies with multi-layer compacted cells were similar to those of mouse and rat ESCs (Fig. 4B). At 11 days to 12 days after the initial induction, EGFP expression could be detected in several iPS-like colonies (approximately 0.5% of all colonies) (Fig. 4C). The iPS-like colonies that express EGFP were harvested for further culture. EGFP expression rapidly weakened and completely disappeared at the first or second passage. These colonies can proliferate and maintain iPSC morphology in both BL and 3i media. Twenty three colonies in BL (hLIF plus bFGF) medium and 31 colonies in 3i (hLIF plus 3i) medium were passaged, and 13 colonies in BL medium and 10 colonies in 3i medium can be cultured for as long as 15 passages with normal karyotypes (Fig. 4D). Immunofluorescence demonstrated that most of the colonies without EGFP expressed Oct4 (Fig. 4E). RT-PCR analysis showed that the detected Oct4 expression was derived from exogenous Oct4 (human Oct4) instead of endogenous Oct4. These results indicated that the pluripotency of Rb-iPSCs was maintained by exogenous Oct4 and not by endogenous Oct4 (Fig. 4F).


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)

Generation of Rb-iPSCs from transgenic Rb-NSCs.(A) Experimental outline for generating Rb-iPSCs from Rb-NSCs. (B) The morphology of NSCs changed to multi-layer compacted clones after 11 days of induction using human transcription factors Oct4, Klf4, Sox2 and c-Myc. (C) EGFP activated in several Rb-iPS colonies in 3i medium (hLIF plus 3i) and BL medium (hLIF plus bFGF). (D) Rb-iPSCs (Rb-iPSC bl13 cultured in BL medium and Rb-iPSC i5 cultured in 3i medium) maintained a normal karyotype at 15 passages. (E) Immunostaining assay showed the expressed Oct4 in Rb-iPSCs. (F) RT-PCR result demonstrating that endogenous Oct4 was not expressed in all the proliferated colonies and that exogenous Oct4 was continuously expressed in Rb-iPSC colonies. (G) Expressed markers of the three germ layers after Rb-iPSCs differentiated in vitro. Tuj1 (ectoderm); AFP (endoderm); and smooth muscle actin (mesoderm). Scale bars = 50 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0109728-g004: Generation of Rb-iPSCs from transgenic Rb-NSCs.(A) Experimental outline for generating Rb-iPSCs from Rb-NSCs. (B) The morphology of NSCs changed to multi-layer compacted clones after 11 days of induction using human transcription factors Oct4, Klf4, Sox2 and c-Myc. (C) EGFP activated in several Rb-iPS colonies in 3i medium (hLIF plus 3i) and BL medium (hLIF plus bFGF). (D) Rb-iPSCs (Rb-iPSC bl13 cultured in BL medium and Rb-iPSC i5 cultured in 3i medium) maintained a normal karyotype at 15 passages. (E) Immunostaining assay showed the expressed Oct4 in Rb-iPSCs. (F) RT-PCR result demonstrating that endogenous Oct4 was not expressed in all the proliferated colonies and that exogenous Oct4 was continuously expressed in Rb-iPSC colonies. (G) Expressed markers of the three germ layers after Rb-iPSCs differentiated in vitro. Tuj1 (ectoderm); AFP (endoderm); and smooth muscle actin (mesoderm). Scale bars = 50 µm.
Mentions: We used Rb-NSCs isolated from the Oct4-EGFP transgenic fetuses to tentatively generate Rb-iPSCs through the infection of lentiviral vectors that encode four human transcription factors (Oct4, Sox2, Klf4 and c-Myc) (Fig. 4A). The cellular morphology changed at 4 days after infection, and iPS-like colonies were first observed after 8 days. The morphological characteristics of iPS-like colonies with multi-layer compacted cells were similar to those of mouse and rat ESCs (Fig. 4B). At 11 days to 12 days after the initial induction, EGFP expression could be detected in several iPS-like colonies (approximately 0.5% of all colonies) (Fig. 4C). The iPS-like colonies that express EGFP were harvested for further culture. EGFP expression rapidly weakened and completely disappeared at the first or second passage. These colonies can proliferate and maintain iPSC morphology in both BL and 3i media. Twenty three colonies in BL (hLIF plus bFGF) medium and 31 colonies in 3i (hLIF plus 3i) medium were passaged, and 13 colonies in BL medium and 10 colonies in 3i medium can be cultured for as long as 15 passages with normal karyotypes (Fig. 4D). Immunofluorescence demonstrated that most of the colonies without EGFP expressed Oct4 (Fig. 4E). RT-PCR analysis showed that the detected Oct4 expression was derived from exogenous Oct4 (human Oct4) instead of endogenous Oct4. These results indicated that the pluripotency of Rb-iPSCs was maintained by exogenous Oct4 and not by endogenous Oct4 (Fig. 4F).

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