Figure 1.
ZFN-mediated genome editing in GS cells.
(A) Neon transfection (1200/30/1) was used to transfect 3×10e5 cells with 1.0 µg of em-GFP plasmid DNA (pCDNA6.2/emGFP) or 1.0 µg of capped and poly-adenylated mRNA coding for pmaxGFP and transfection efficiency was quantified by flow cytometry three days after transfection. Lipofectamine-2000 was used to transfect the same ratio of cells:DNA or cells:mRNA as in the Neon experiment. The mean and standard deviation of percentage of GFP+ cells from three experiments are shown. (*p<0.05, **p<0.01,***p<0.001, Student T test). (B) Schematic depicting the two plasmids used in genome editing experiments. The donor DNA (“37GFP”; plasmid BE356) contains a fragment of the GFP coding sequence lacking the first 37 nucleotides and serves as a donor template. “Ubc-ZFN1-T2A-ZFN2” (plasmid M500) contains a bicistronic expression cassette with a human Ubiquitin C promoter driving expression of two ZFNs directed to a recognition site in the GFP gene and separated by a T2A skip sequence. GS cell lines were derived from mice carrying a mutated GFP gene, with a 85 nucleotide stop codon and frame shift insertion (labeled “STOP”), introduced into the ROSA26 locus by standard knockin technology [25]. (C) 0.8 µg of Ubc-ZFN1-T2A-ZFN2 (M500) plasmid or 0.8 µg each of synthesized mRNA ZFN1 and ZFN2, together with 2.4 µg donor plasmid (BE356), were transfected (1400/20/1) into MPG6 cells on day 1 and genome editing events were quantified on day 5 or 7 (data pooled). Histogram shows mean +/- standard error mean. The dot plot shows sample results of a single transfection of donor DNA and ZFN mRNAs. (D) GFP fluorescence (left) or corresponding transmitted light image in GT59 cells following two sorts to enrich for GFP+ cells. Bar represents 50 microns. (E) Chromatogram showing corrected GFP gene sequence of PCR amplified genomic DNA from GT59 cells. The ZFN recognition sites are indicated by boxes and the site in which the mutation was replaced by donor DNA is indicated by a line.
Figure 2.
Retention of the spermatogonial phenotype following gene correction.
Immunostaining was performed on gene-corrected GT59 (left) and GT65 cells (right): DAZL, a germ cell specific marker; GFRA1, POU5F1, ETV5, CDH1, and SOHLH1, markers of undifferentiated spermatogonia. Additionally, GT59 and GT65 cells were treated with the differentiation factor, retinoic acid (1 µM) or a vehicle control and then immunostained to examine levels of ZBTB16, a marker of undifferentiated spermatogonia. Bar represents 50 microns.
Figure 3.
Colonization by gene-corrected GS cells following testicular transplantation.
(A) Whole mounted squash preparation of seminiferous tubules depicting a seminiferous tubule (arrows) extensively colonized by gene-corrected GT59 SSCs two months following transplantation into a KitW-v/KitW sterile pup testis. (B) Non-transplanted control. (C) Whole mounted squash preparation of seminiferous tubules depicting a colony of GT65 SSCs two months following transplantation into a busulfan treated adult testis. Visualization of GT65 cells was facilitated by modification with Histone-GFP lentivirus prior to transplant. (A–C) Large arrowheads indicate GFP+ colonies and small arrows indicate autofluorescence in nearby tubules. Bar = 100 microns. (C′) Higher magnification image of the boxed area in (C). Bar = 50 microns. (D–G) Immunostaining with anti-GFP antibody (E, G, E′) or DAPI staining (D, F, D′, F′) of a cryosection of a GT59 colony 6 months following transplantation into KitW-v/KitW pup testis (D, E, D′, E′) or non-transplanted control testes (F, G, F′). Boxed area in D corresponds to the higher magnification view in D′ and E′. Triangles indicate donor-derived GFP+ cells. Boxed area in F corresponds to the higher magnification view in F′ and depicts the “Sertoli Cell Only” phenotype of non-transplanted KitW-v/KitW testes. The Sertoli cells are indicated by open triangles. Bar = 25 microns.