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. 2011 Dec 21:7:100.
doi: 10.1186/1744-8069-7-100.

Temporal control of gene deletion in sensory ganglia using a tamoxifen-inducible Advillin-Cre-ERT2 recombinase mouse

Joanne Lau  1 , Michael S Minett, Jing Zhao, Ulla Dennehy, Fan Wang, John N Wood, Yury D Bogdanov
Affiliations

Temporal control of gene deletion in sensory ganglia using a tamoxifen-inducible Advillin-Cre-ERT2 recombinase mouse

Joanne Lau et al. Mol Pain. .

Abstract

Background: Tissue-specific gene deletion has proved informative in the analysis of pain pathways. Advillin has been shown to be a pan-neuronal marker of spinal and cranial sensory ganglia. We generated BAC transgenic mice using the Advillin promoter to drive a tamoxifen-inducible CreERT2 recombinase construct in order to be able to delete genes in adult animals. We used a floxed stop ROSA26LacZ reporter mouse to examine functional Cre expression, and analysed the behaviour of mice expressing Cre recombinase.

Results: We used recombineering to introduce a CreERT2 cassette in place of exon 2 of the Advillin gene into a BAC clone (RPCI23-424F19) containing the 5' region of the Advillin gene. Transgenic mice were generated using pronuclear injection. The resulting AvCreERT2 transgenic mice showed a highly specific expression pattern of Cre activity after tamoxifen induction. Recombinase activity was confined to sensory neurons and no expression was found in other organs. Less than 1% of neurons showed Cre expression in the absence of tamoxifen treatment. Five-day intraperitoneal treatment with tamoxifen (2 mg per day) induced Cre recombination events in ≈90% of neurons in dorsal root and cranial ganglia. Cell counts of dorsal root ganglia (DRG) from transgenic animals with or without tamoxifen treatment showed no neuronal cell loss. Sensory neurons in culture showed ≈70% induction after 3 days treatment with tamoxifen. Behavioural tests showed no differences between wildtype, AvCreERT2 and tamoxifen-treated animals in terms of motor function, responses to light touch and noxious pressure, thermal thresholds as well as responses to inflammatory agents.

Conclusions: Our results suggest that the inducible pan-DRG AvCreERT2 deleter mouse strain is a useful tool for studying the role of individual genes in adult sensory neuron function. The pain phenotype of the Cre-induced animal is normal; therefore any alterations in pain processing can be unambiguously attributed to loss of the targeted gene.

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Figures

Figure 1
Figure 1
Generation of tamoxifen inducible Advillin Cre mouse. A - Diagram of the wt Advillin allele and the targeting construct. CreERT2 is inserted downstream of the initiation codon of Advillin. The construct also contained a Kanamycin selection gene that was excised from the BAC by Flp-recombination. B - Primer validation for screening of transgenic animals. AF - Advillin forward primer, AR - Advillin reverse primer, CF - Cre forward primer, CR - Cre reverse primer. The expected sizes of the PCR fragments are indicated. C - Southern blot with tail genomic DNA digested with HindIII and hybridized with the 5'homology arm probe. The bands for wildtype gene (wt) and transgene (tg) are indicated.
Figure 2
Figure 2
Tamoxifen induces recombination in the DRG of adult AvCreERT2 mouse. Animals were injected (ip) for 5 consecutive days (2 mg per day). A - X-gal staining of adult DRG neurons from induced and un-induced mice. Neutral red was used for counterstaining. B - Quantification of recombination events in DRG of untreated and tamoxifen-treated animals. Data are presented as mean ± SEM. Statistical analysis - unpaired T-test, p ≤ 0.01. Scale bars = 40 μm.
Figure 3
Figure 3
Co-localization of X-gal staining with peripherin and NF200 positive neurons in the dorsal root ganglia of the adult AvCreERT2 mice. A - X-gal (transmitted light) and B. - Anti-peripherin (green) and anti-NF200 staining (red). C - Tamoxifen treatment does not alter the composition of DRG. White bars - wt, Grey bars - uninjected AvCreERT2 animals, Black bars - AvCreERT2 animals injected with tamoxifen (2 mg per day, 5 days). D - Quantification of the total number of neurons per DRG of wildtype and AvCreERT2 mice (untreated and injected with tamoxifen). Data are presented as mean ± SEM. Scale bars = 40 μm.
Figure 4
Figure 4
Tamoxifen induces Cre-expression during embryonic development. A - E18.5 AvCreERT2-positive embryos from tamoxifen treated pregnant females (2 mg per day, 5 days). B - E18.5 AvCreERT2-positive embryos from vehicle treated pregnant females.
Figure 5
Figure 5
4-OHT induces Cre-expression in cell culture of neurons from dorsal root ganglia of AvCreERT2 mice. A - X-Gal, anti-peripherin (green) and anti-NF200 (red) staining of 4-OHT treated and uninduced cell cultures from DRG. Arrows indicate X-gal stained neurons. Note the punctuate nature of X-gal staining. B - Quantification of recombination events in untreated and 4-OHT treated cultures of neurons from DRGs. Data are presented as mean ± SEM. Statistical analysis - unpaired T-test, p ≤ 0.01. Scale bars = 40 μm.
Figure 6
Figure 6
AvCreERT2 expression or tamoxifen treatment (10 mg) does not affect acute nociceptive responses. A - Behavioural responses of AvCreERT2 and littermate mice to Aa) Rotarod (AvCreERT2 N = 13, littermate N = 11), Ab) von Frey (AvCreERT2 N = 13, littermate N = 11), Ac) Randall-Selitto (AvCreERT2 N = 8, littermate N = 8), Ad) Hargreaves' (AvCreERT2 N = 13, littermate N = 11), Ae) (AvCreERT2 N = 7, littermate N = 8) Af) Acetone (AvCreERT2 N = 8, littermate N = 8). No significant difference was found (t-test). Data are expressed as mean ± SEM. B - C57BL/6 mice received a five-day course of tamoxifen (2 mg/0.2 ml per day), or vehicle (0.2 ml per day). Responses to Ba) Rotarod (tamoxifen N = 12, vehicle N = 11) Bb) von Frey (tamoxifen N = 12, vehicle N = 11). Bc) Randall-Selitto (tamoxifen N = 6, vehicle N = 5) Bd) Hargreaves' (tamoxifen N = 12, vehicle N = 11) Be) 50 & 55°C Hot plate (tamoxifen N = 12, vehicle N = 11) Bf) Acetone (tamoxifen N = 12, vehicle N = 11). Animals were examined 7 days prior to tamoxifen treatment then 7 and 56 days after. No significant differences were found (Two-way repeated measures ANOVA). Data are expressed as mean ± SEM.
Figure 7
Figure 7
AvCreERT2 expression or tamoxifen (10 mg) treatment does not affect inflammatory nociceptive responses. A - Behavioural responses of AvCreERT2 and littermate mice to inflammatory pain tests Aa) Pain behaviour of AvCreERT2 (N = 6) & littermate (N = 6) mice after intraplantar injection of 20 μl of 5% formalin. Ab) Time spent licking/biting the injected hindpaw in phase I (1-10 min) and phase II (10-60 min) (AvCreERT2 N = 6, littermate N = 6), Ac) Mechanical allodynia following intraplantar injection of 20 μl of CFA (AvCreERT2 N = 7, littermate N = 6), Ad) Thermal hyperalgesia after intraplantar injection of 20 μl of CFA (AvCreERT2 N = 7, littermate N = 6). No significant difference was found (Two-way repeated measures ANOVA). Data are expressed as mean ± SEM. B - Behavioural responses of C57BL/6 mice injected with a five-day course of tamoxifen (2 mg/0.2 ml per day), or vehicle (0.2 ml per day) to inflammatory pain tests. Ba) Pain behavior of tamoxifen-treated (N = 6) & vehicle-treated (N = 6) mice after intraplantar injection of 20 μl of 5% formalin. Bb) Time spent licking/biting the injected hindpaw in phase I (1-10 min) and phase II (10 -60 min) (tamoxifen N = 6, vehicle N = 6), Bc) Mechanical allodynia following intraplantar injection of 20 μl of CFA (tamoxifen N = 6, vehicle N = 6), Bd) Thermal hyperalgesia after intraplantar injection of 20 μl of CFA (tamoxifen N = 6, vehicle N = 6). Animals were examined 7 days following tamoxifen/vehicle treatment. No significant differences were found (Two-way repeated measures ANOVA). Data are expressed as mean ± SEM.
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