Expression level can be increased when selectable marker is removed.

<div><p>(<b>A</b>) Diagram of orientation of constructs in cell lines.</p> <p>(<b>B</b>) A2B2 and A2B2<i>Δ</i> cells induced with doxycycline (<b>i</b>) A2B2 phase contrast (<b>ii</b>) A2B2 EGFP expression (<b>iii</b>) A2B2<i>Δ</i> phase contrast (<b>iv</b>) A2B2<i>Δ</i> EGFP expression</p> <p>(<b>C</b>) Graph of expression levels in induced and uninduced cell lines quantitated by fluorimetry.</p> <p>Expression of EGFP is higher when these cell lines have lost the selectable marker cassette (<i>F</i>(1,40) = 77.25, <i>p</i><0.0001).</p> <p>The orientation of the TRE EGFP makes no difference to expression level (<i>F</i>(1,40) = 1.14, <i>p</i> = 0.2910).</p> <p>Each point is an average of three measurements each from two independently targeted cell lines.</p> <p>Error bars denote standard deviations. Asterisks indicate statistically significant differences.</p></div

Expression level is decreased when selectable marker is removed and promoter is in same orientation as ROSA26 promoter.

<div><p>(<b>A</b>) Diagram of orientation of constructs in cell lines.</p> <p>(<b>B</b>) Α1Β1 ανδ Α1Β1Δ cells induced with doxycycline (<b>i</b>) A1B1 phase contrast (<b>ii</b>) A1B1 EGFP expression (<b>iii</b>) A1B1<i>Δ</i> phase contrast (<b>iv</b>) A1B1<i>Δ</i> EGFP expression (<b>C</b>) Graph of expression levels in induced and uninduced cell lines quantitated by fluorimetry.</p> <p>Expression of EGFP is lower when the selectable marker is removed from the A1 cell lines (<i>F</i>(1,40) = 51.77, <i>p</i><0.001).</p> <p>Again the orientation of the TRE-EGFP makes no significant difference to the expression levels observed (<i>F</i>(1,40) = 0.05, <i>p</i> = 0.8295).</p> <p>Each point is an average of three measurements each from two independently targeted cell lines.</p> <p>Error bars denote standard deviations.</p> <p>Asterisks indicate statistically significant differences.</p></div

Targeting strategy for introducing to ROSA26locus.

<div><p>(<b>A</b>). Diagram of targeting constructs to introduce Dox responsive transgenes into locus.</p> <p>A1 and A2 introduce the same activator transgene in opposite orientations.</p> <p>B1 and B2 introduce the same responder transgene in opposite orientations.</p> <p>(<b>B</b>) Four different cell lines were produced with the activator and responder transgenes in different orientations (<b>C</b>) Southern blots probed with 5′ and 3′ flanking probes produce the correct band sizes to demonstrate appropriate targeting of constructs.</p></div

Dox regulated gene expression works appropriately at the ROSA26 locus.

<div><p>(<b>A</b>) EGFP expression induced in A1B1 cells by addition of dox to a final concentration of 1 µgml⁻¹.</p> <p>(<b>i</b>) and (<b>iii</b>) Phase contrast.</p> <p>(<b>ii</b>) and (<b>iv</b>) EGFP fluorescence (<b>B</b>) EGFP expression in single targeted and double targeted cell lines in reponse to 1 µgml⁻¹ dox.</p> <p>Induction of EGFP is only seen in the A1B1 cells (F(2,30) = 1167.61, p<0.0001).</p> <p>Each point is an average of three measurements each from two independently targeted cell lines.</p> <p>Error bars denote standard deviations.</p> <p>Asterisks indicate statistically significant differences.</p> <p>(<b>C</b>) Dose response curve EGFP expression in A1B1 cells in response to dox.</p></div

Leydig and Sertoli cells are not affected by Taz deficiency.

<p>Immunohistology sections of SV129P2 wild type (A,B, G,I) and Taz^<b>Neo</b> testis tubules (B,D, H and J) showing the staining of Leydig cell marker 3<i>β</i>-HSD (Hsd3b6) (A,D), sertoli cell nucleus Wt1 (B,D). Recipient Sertoli cell non-contribution in Taz^<b>Neo</b> testis tubules (E, F): low chimera section showing wild type and defective tubules as stained with Hprt (E) and adjacent section stained for Tubb3 showing sertoli cell cytoplasm (F). Taz^<b>Neo</b> semiferous tubules showed increased cell death. Caspase-3 staining of sections of SV129P2 wild type (G) and Taz^<b>Neo</b> (H) showing the occurrence of cell death (brown nuclear staining) in Taz^<b>Neo</b> seminiferous tubules. Ki67 immunostaining of the control tubule is found restricted to dividing spermatogonia in the basal region of the tubules (I) as Taz^<b>Neo</b> tubules display positive staining through the tubule (J). Scale bar: 250<i>μ</i>m.</p

Taz deficient seminiferous tubules showed increased DNA damage due to higher L1 retrotransposon activity.

<p>A, B: immunostaining of P16 nuclear spread from control (A) or Taz^<b>Neo</b> testis (B) using anti-<i>γ</i>H2ax (red) antibody. Dapi nuclear stain (blue). Scale bar 50<i>μ</i>m. C-J: Immunostaining of adult testis sections of control (C,E,G,I) and Taz^<b>Neo</b> testis (D,F,H,J) with anti-<i>γ</i>H2ax (C-F) or anti-Orf1-Line1 (G-J) antibody. White arrows: sex bodies seen on germ cells reaching Pachytene stage (E). Black arrow heads indicating typical punctuating staining of DNA damage (F). Black arrows: Orf1 Line1 nuclear staining (J). Scale bars: 250<i>μ</i>m.</p

Taz^Neo spermatocytes do not differentiate to the round spermatid stage.

<p>Immunohistology sections of SV129P2 wild type (A,B,E,F,G) and TAZ^<b>Neo</b> testis (C,D,H, I and J) showing the staining of sperm germ cells marker, Dazl (A,C), Primordial germ cell marker, Vasa (B,D), mature germ cell markers Actl7B (E,H), Hook1 (F,I) and Pgk2 (G,J). Double arrows on each of the wild-type sections (A,B,E,F,G) indicate the zone of differentiating spermatozoa absent in the Taz^<b>Neo</b> tubules. Scale bar: 250<i>μ</i>m</p

Taz^Neo ES cells don’t express spermiogenesis markers when differentiated in vitro.

<p>A: Top Panel: Scheme of the introduction of the flox stop Dazl construct at the HPRT locus before and after Cre deletion and recombination at the lox P sites (black arrow head). B: RT-PCR of Taz and various sperm differentiation markers (Prm1, Tnp2, Dmc-1, Sycp-3, Sycp-1) with cDNA extracted from HM1 parental ES clones or Taz^<b>Neo</b> ES cells before or after 19 days differentiation. C: Western blotting of wild type or Taz deficient ES cell clone protein extract with or without 19 days differentiation for Dazl, Sycp-3 or Vasa. <i>β</i>-actin is used to show equal loading.</p

Taz deficient chimeras fail to produce mature sperm.

<p>Left panels: Histology sections of control (A,C,E,G) and Taz^<b>Neo</b> tissue (B,D,F,H) showing the epididymis (A,B), P8 (C,D), P16 (E,F) and adult testis tubule (G,H). Sections through the epididymis show a complete lack of differentiated sperm in the lumen of the Taz^<b>Neo</b> epididymis (B). White arrowhead: multinuclear cells. Black arrows: vacuoles. Scale bars: 250<i>μ</i>m. Right panels (I, J, K) show the high contribution to chimerism of Taz deficient cells in chimera testis: HPRT staining of wild type SV129P2 adult testis (I), of a low level of chimerism testis (J) (defective tubule harbouring numerous vacuoles is negative for HPRT staining*) and of a high level of chimerism testis (K). Scale bar: 250<i>μ</i>m</p

Taz^Neo germ cell differentiation fails before reaching the Pachytene stage of meiosis I.

<p>Sycp-3 immunostaining of nuclear spread from control (left) and Taz deficient P16 spermatocytes assessing the different stages of the Meiosis I prophase I during spermatocytes differentiation (leptotene, zygotene, pachytene). Scale bars: 50<i>μ</i>m. Proportion of cells in various prophase I stages in control or Taz deficient spermatocytes (lower right panels).</p