egfp reporter gene (enhanced green fluorescent protein)  (Addgene inc)

Journal: Biology of Reproduction

Article Title: DDX4- EGFP transgenic rat model for the study of germline development and spermatogenesis †

doi: 10.1095/biolreprod.116.142828

Figure Lengend Snippet: Egfp mRNA expression in testis and ovary compared to somatic tissues in DDX4-EGFP transgenic rats. Fold changes of Egfp mRNA levels (2 −ΔΔCt ) in selected organs are shown. The deltaCt ( Egfp Ct – Actb ( β-actin ) Ct) was computed for each tissue on each animal. Repeated-measures ANOVA was used to determine whether the deltaCt values were significantly different across tissues. All tissues were compared to the testis (reference group) by calculating delta-delta Ct values (dCt(tissue) –dCt(testis)). Egfp expression in testis and ovary was compared between wildtype and transgenic animals using two-tailed Student's t -test. P -values less than 0.05 were considered statistically significant and are denoted by different letters.

Article Snippet: We used the Ddx4 (formerly known as Vasa) gene promoter [ ] to drive enhanced green fluorescence protein ( Egfp ) reporter gene expression in germ cells of Sprague-Dawley (SD) rats.

Techniques: Expressing, Transgenic Assay, Two Tailed Test

Journal: Biology of Reproduction

Article Title: DDX4- EGFP transgenic rat model for the study of germline development and spermatogenesis †

doi: 10.1095/biolreprod.116.142828

Figure Lengend Snippet: Live imaging evaluation of endogenous EGFP expression in selected organs of DDX4-EGFP transgenic rats. Representative bright-field (left) and fluorescent (right) images of selected organs from DDX4-EGFP transgenic rats are shown. Wildtype testes and ovaries served as negative controls. Strong endogenous EGFP expression was observed in transgenic testes. In transgenic ovaries, EGFP was observed in oocytes that are visible as small round green fluorescent dots. Somatic tissues did not express EGFP. Scale bar = 1 mm.

Article Snippet: We used the Ddx4 (formerly known as Vasa) gene promoter [ ] to drive enhanced green fluorescence protein ( Egfp ) reporter gene expression in germ cells of Sprague-Dawley (SD) rats.

Techniques: Imaging, Expressing, Transgenic Assay

Journal: Biology of Reproduction

Article Title: DDX4- EGFP transgenic rat model for the study of germline development and spermatogenesis †

doi: 10.1095/biolreprod.116.142828

Figure Lengend Snippet: EGFP immunohistochemistry of various tissues from F3 offspring. Paraffin embedded tissue sections were stained with anti-EGFP antibody. Only germ cells in DDX4-EGFP testes (A) and ovaries (C) were EGFP positive. No staining was evident in wildtype testis (B) and ovary (D) sections, or any of the other organs that were evaluated (E–L). Sections were not counterstained with DAPI to allow observation of the different levels of autofluorescence in intestine, spleen, lung, and kidney tissue. Scale bars = 10 μm.

Article Snippet: We used the Ddx4 (formerly known as Vasa) gene promoter [ ] to drive enhanced green fluorescence protein ( Egfp ) reporter gene expression in germ cells of Sprague-Dawley (SD) rats.

Techniques: Immunohistochemistry, Staining

Journal: Biology of Reproduction

Article Title: DDX4- EGFP transgenic rat model for the study of germline development and spermatogenesis †

doi: 10.1095/biolreprod.116.142828

Figure Lengend Snippet: Mixed EGFP expression in oocytes of DDX4-EGFP transgenic rats. EGFP expression in ovaries was determined by coimmunofluorescence staining for DDX4 and EGFP in paraffin-embedded ovarian sections. (A–C) DDX4-positive, EGFP-positive rat oocyte. (D–F) Not all oocytes in DDX4-EGFP ovaries expressed EGFP. Panel E shows the lack of EGFP expression in an oocytes marked by DDX4 staining. (G–I) DDX4 expression is not consistent in all oocytes from 4-week-old rats as shown in panel G. EGFP was expressed in a DDX4-negative oocyte. Scale bars = 10 μm. (J–L) EGFP was not detectable in the majority of early (primordial/primary) DDX4-positive follicles (Arrows). Scale bar = 30 μm. Quantification of the DDX4 and EGFP costaining is represented in M.

Article Snippet: We used the Ddx4 (formerly known as Vasa) gene promoter [ ] to drive enhanced green fluorescence protein ( Egfp ) reporter gene expression in germ cells of Sprague-Dawley (SD) rats.

Techniques: Expressing, Transgenic Assay, Staining

Journal: Biology of Reproduction

Article Title: DDX4- EGFP transgenic rat model for the study of germline development and spermatogenesis †

doi: 10.1095/biolreprod.116.142828

Figure Lengend Snippet: EGFP is expressed in germ cells throughout germ lineage development and spermatogenesis in postnatal transgenic DDX4-EGFP testes. Germ cells are identified by positive DDX4 staining at 1, 8, 14, and 28 dpp, and in adult testes (left column top to bottom). EGFP was consistently expressed in all DDX4-positive germ cells during postnatal development and during spermatogenesis (center column top to bottom), as confirmed in merged images (right column top to bottom). Scale bar = 50 μm.

Article Snippet: We used the Ddx4 (formerly known as Vasa) gene promoter [ ] to drive enhanced green fluorescence protein ( Egfp ) reporter gene expression in germ cells of Sprague-Dawley (SD) rats.

Techniques: Transgenic Assay, Staining

Journal: Biology of Reproduction

Article Title: DDX4- EGFP transgenic rat model for the study of germline development and spermatogenesis †

doi: 10.1095/biolreprod.116.142828

Figure Lengend Snippet: Transplanted SSCs from DDX4-EGFP rats produce green fluorescent colonies of spermatogenesis. (A) Single-cell suspensions from 14-day-old DDX4-EGFP testes were stained with anti-DDX4 antibody to identify germ cells, and anti-EGFP antibody to detect transgene expressing germ cells. All germ cells expressed EGFP. (B) The same single-cell suspension was costained with anti-SALL4 antibody to mark undifferentiated spermatogonia including SSCs. While most SALL4-positive spermatogonia at this age expressed EGFP, SALL4-negative/EGFP-positive germ cells were also observed (white arrowheads). Scale bars = 10 μm. (C) The same single-cell suspension as shown in A and B was transplanted into busulfan-treated infertile nude mouse recipient testes. Three months after transplantation, EGFP-positive colonies of donor-derived spermatogenesis could be observed, confirming the expression of the reporter gene in SSCs.

Article Snippet: We used the Ddx4 (formerly known as Vasa) gene promoter [ ] to drive enhanced green fluorescence protein ( Egfp ) reporter gene expression in germ cells of Sprague-Dawley (SD) rats.

Techniques: Staining, Expressing, Suspension, Transplantation Assay, Derivative Assay

Journal: Biology of Reproduction

Article Title: DDX4- EGFP transgenic rat model for the study of germline development and spermatogenesis †

doi: 10.1095/biolreprod.116.142828

Figure Lengend Snippet: FACS isolation of DDX4-EGFP-positive spermatogonia for long-term rat SSC culture. (A) Sorting gates for separating EGFP-positive cells from EGFP-negative cells. Sorting of PND 10 testicular cells is shown as a representative example. (B) Transplantation of negative fractions into infertile recipient mouse testes did not result in colonies of spermatogenesis. (C) Abundant green fluorescent colonies of spermatogenesis were obtained when transplanting the same cell number of EGFP-positive sorted fractions into infertile recipient testes. Scale bar in B and C is 1 mm. (D–F) EGFP-positive germ cells isolated from PND 10 rat testes were cultured on STO feeder cells and maintained endogenous EGFP expression. Scale bar = 50 μm. (G–I) Long-term culture of rSSCs for 12 weeks was established that maintains DDX4 expression (germ cell marker), and SALL4 and ZBTB16 (two established spermatogonia marker). Scale bars = 50 μm.

Article Snippet: We used the Ddx4 (formerly known as Vasa) gene promoter [ ] to drive enhanced green fluorescence protein ( Egfp ) reporter gene expression in germ cells of Sprague-Dawley (SD) rats.

Techniques: Isolation, Transplantation Assay, Cell Culture, Expressing, Marker