Time-dependent Conditional Knockout Mouse Models (Inducible)
A time-dependent conditional Knockout mouse defines an inducible animal model in which a gene of interest is "floxed," thus temporally controllable at a given time-point in embryonic, post-natal or adult animals.
After an additional breeding step with a Cre-ERt2 deleter mouse line, the conditional Knockout is temporally triggered by external inducer-agents, most often small molecules such as tamoxifen or tetracycline.
Typical applications for time-dependent conditional Knockout mouse models
For academic research:
For bio-pharmaceutical research & development:
Strengths and limitations of time-dependent conditional Knockout mouse models
- Very flexible: easy switch to study time-dependent KOs in different tissues
- Large resource of deleter mice (Cre or FLP) enabling an almost infinite combination of time-dependent KOs in different tissues
- High physiological relevancy of the scientific data obtained from the model
- Many deletion-inducible lines (thousands of Cre-FLP mouse lines...) enabling almost infinite specificity
- Expression levels dependent on the dose of the agent administered
- Differential penetration of trigger compound into tissue
- Availability and background of Cre inducer lines
- Adding the loxP sites risks modification or disruption of splicing regulation (ESS ESE); possible impact on overlapping and neighboring genes
→ Need careful analysis of the placement of loxP sites
- Tetracycline-inducible systems can be leaky
→ Temporal control can be achieved by using a fusion between Cre and a mutated form of the ligand-binding domain of the estrogen receptor which only binds tamoxifen (ERt and ERt2). This inactive Cre-ERt2 fusion is activated upon tamoxifen (or 4-hydroxytamoxifen) administration.
Case studies and publications on our time-dependent conditional Knockout mouse models
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 Alkbh4L/L (flanked by loxP) mice crossed with CreEsr transgenic mice to create the inducible Knockout genotype Alkbh4L/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 Alkbh4L/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 Alkbh4Δ/Δ mice after 2 weeks of tamoxifen treatment detected by Western blot.
Figure 2. Loss of Alkbh4 reduces the size of testis
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).
Figure 3. Stage-specific arrest of spermatogenesis in Alkbh4Δ/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 (Alkbh4L/L) and Alkbh4Δ/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 Alkbh4Δ/Δ mice.
C) Mean cell density at selected stages (I, VIII, IX) of spermatogenesis in Alkbh4Δ/Δ mice indicates depletion of cells at pachynema and all subsequent stages.
Lima WF, Murray HM, Damle SS, Hart CE, Hung G, De Hoyos CL, Liang XH, Crooke ST.
Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function.
Nucleic Acids Res. 2016 Jun 20.
Frye M, Dierkes M, Küppers V, Vockel M, Tomm J, Zeuschner D, Rossaint J, Zarbock A, Koh GY, Peters K, Nottebaum AF, Vestweber D.
Interfering with VE-PTP stabilizes endothelial junctions in vivo via Tie-2 in the absence of VE-cadherin.
J Exp Med. 2015 Dec 14.
Birdsey GM, Shah AV, Dufton N, Reynolds LE, Osuna Almagro L, Yang Y, Aspalter IM, Khan ST, Mason JC, Dejana E, Göttgens B, Hodivala-Dilke K, Gerhardt H, Adams RH, Randi AM.
The Endothelial Transcription Factor ERG Promotes Vascular Stability and Growth through Wnt/β-Catenin Signaling.
Dev Cell. 2015 Jan 20.
Nilsen A, Fusser M, Greggains G, Fedorcsak P, Klungland A
ALKBH4 Depletion in Mice Leads to Spermatogenic Defects.
PLoS One. 2014 Aug 25.
Liu S, Thompson K, Leask A.
CCN2 expression by fibroblasts is not required for cutaneous tissue repair.
Wound Repair Regen. 2014 Jan.
Adolph TE, Tomczak MF, Niederreiter L, Ko HJ, Böck J, Martinez-Naves E, Glickman JN, Tschurtschenthaler M, Hartwig J, Hosomi S, Flak MB, Cusick JL, Kohno K, Iwawaki T, Billmann-Born S, Raine T, Bharti R, Lucius R, Kweon MN, Marciniak SJ, Choi A, Hagen SJ, Schreiber S, Rosenstiel P, Kaser A, Blumberg RS.
Paneth cells as a site of origin for intestinal inflammation.
Nature. 2013 Nov 14.
Goettsch C, Babelova A, Trummer O, Erben RG, Rauner M, Rammelt S, Weissmann N, Weinberger V, Benkhoff S, Kampschulte M, Obermayer-Pietsch B, Hofbauer LC, Brandes RP, Schröder K.
NADPH oxidase 4 limits bone mass by promoting osteoclastogenesis.
J Clin Invest. 2013 Nov 1.
Hardy AB, Wijesekara N, Genkin I, Prentice KJ, Bhattacharjee A, Kong D, Chimienti F, Wheeler MB.
Effects of high-fat diet feeding on Znt8-null mice: differences between β-cell and global knockout of Znt8.
Am J Physiol Endocrinol Metab. 2012 May.
Liu S, Shi-wen X, Abraham DJ, Leask A.
CCN2 is required for bleomycin-induced skin fibrosis in mice.
Arthritis Rheum. 2011 Jan.
Whelan G, Kreidl E, Wutz G, Egner A, Peters JM, Eichele G.
Cohesin acetyltransferase Esco2 is a cell viability factor and is required for cohesion in pericentric heterochromatin.
EMBO J. 2011 Nov 18.
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