Time-dependent KO mouse

Case study

Model to circumvent embryonic lethality and study male germ cells

‍Nilsen A, et al. ALKBH4 depletion in mice leads to spermatogenic defects. PLoS One. 2014.

ALKBH4 indirectly influences the ability of non-muscular myosin II to bind actin filaments. It modulates fundamental processes including cytokinesis and cell motility, and its depletion is lethal during early preimplantation embryo stage.

Model: Conditional Alkbh4^L/L (flanked by loxP) mice crossed with CreEsr transgenic mice to create the inducible Knockout genotype Alkbh4^L/L CreEsr overcoming the embryonic lethality.

Aim: Investigate the effect of ALKBH4 deficiency in a physiological context, using inducible Alkbh4 Knockout mice.

Results: ALKBH4 is essential for the development of spermatocytes during the prophase of meiosis.

Figure 1. Tamoxifen (TAM) treatment and suppression of Alkbh4.

A) Schematic outline of TAM treatment in of Alkbh4^L/L CreEsr and control mice day 0 to 14 with indicated time-­points for sampling.

B) Cre-­mediated recombination of the loxP-­flanked DNA sequence in selected organs of Alkbh4 mice after 2 weeks of tamoxifen treatment detected by PCR.

C) Depletion of ALKBH4 in whole-­testis extracts of Alkbh4^Δ/Δmice after 2 weeks of tamoxifen treatment detected by Western blot.

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Figure 2. Loss of Alkbh4 reduces the size of testis

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Left panel) Representative testes from control and Alkbh4 mice after tamoxifen treatment for one week or without treatment (0-1 week), and after deletion of Alkbh4 with treatment for 2 weeks.

Right panel) Average testis/body weight ratio of 4-5-week-old mice before and after 1 week of treatment with tamoxifen (0-1 week), and 6-week-old mice treated with tamoxifen for 2 weeks (0-1 week).

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Figure 3. Stage­-specific arrest of spermatogenesis in Alkbh4^Δ/Δ mice.

A) Immunofluorescence labeling of histological sections for anti-γH2A.X (red) with DAPI (blue) counterstain, where Sertoli cells (Se), leptotene (L) and pachytene (P) spermatocytes and elongating spermatids (S9) can be distinguished.

B) Mean cell density per cross­-section of stage IX (n>10 tubuli per histological section, total of 6 sections) from control (Alkbh4^L/L) and Alkbh4^Δ/Δ mice treated with tamoxifen for 1-2 weeks shows severe depletion of pachytene spermatocytes and S9 elongating spermatids after 2 weeks of tamoxifen treatment in Alkbh4^Δ/Δ mice.

C) Mean cell density at selected stages (I, VIII, IX) of spermatogenesis in Alkbh4^Δ/Δ mice indicates depletion of cells at pachynema and all subsequent stages.