Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Aug 2:8:63.
doi: 10.1186/1471-2202-8-63.

Inducible gene inactivation in neurons of the adult mouse forebrain

Gitta Erdmann  1 , Günther Schütz, Stefan Berger
Affiliations

Inducible gene inactivation in neurons of the adult mouse forebrain

Gitta Erdmann et al. BMC Neurosci. .

Abstract

Background: The analysis of the role of genes in important brain functions like learning, memory and synaptic plasticity requires gene inactivation at the adult stage to exclude developmental effects, adaptive changes or even lethality. In order to achieve temporally controlled somatic mutagenesis, the Cre/loxP-recombination system has been complemented with the tamoxifen-inducible fusion protein consisting of Cre recombinase and the mutated ligand binding domain of the human estrogen receptor (CreERT2). To induce recombination of conditional alleles in neurons of the adult forebrain, we generated a bacterial artificial chromosome-derived transgene expressing the CreERT2 fusion protein under control of the regulatory elements of the CaMKIIalpha gene (CaMKCreERT2 transgene).

Results: We established three mouse lines harboring one, two and four copies of the CaMKCreERT2 transgene. The CaMKCreERT2 transgene displayed reliable and copy number-dependent expression of Cre recombinase specifically in neurons of the adult forebrain. Using Cre reporter mice we show very low background activity of the transgene in absence of the ligand and efficient induction of recombination upon tamoxifen treatment in all three lines. In addition, we demonstrate in mice harboring two conditional glucocorticoid receptor (GR) alleles and the CaMKCreERT2 transgene spatially restricted loss of GR protein expression in neurons of the adult forebrain upon tamoxifen treatment.

Conclusion: This is to our knowledge the first approach allowing highly efficient inducible gene inactivation in neurons of the adult mouse forebrain. This new approach will be a useful tool to dissect the function of specific genes in the adult forebrain. Effects of gene inactivation on pre- and postnatal brain development and compensatory mechanisms elicited by an early onset of gene inactivation can now be excluded.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Copy number-dependent transgene expression and tamoxifen-dependent nuclear translocation of the CreERT2-fusion protein in neurons of the adult hippocampus. (A) The cassette encoding the CreERT2-fusion protein was inserted in frame at the ATG of the CaMKIIα gene present on a large DNA fragment of a BAC vector containing mouse genomic DNA by homologous recombination in bacteria. The genomic insert of the BAC vector contained a 43 kb 5'-upstream and a 100 kb 3'-downstream region of the CaMKIIα gene. (B-G) Immunohistochemical staining of pyramidal neurons in the hippocampal CA1 region of CaMKCreERT2 transgenic animals with different transgene copy numbers using an antibody against Cre recombinase revealed non-nuclear localization of the Cre recombinase in vehicle-injected animals (B, D, F) and nuclear localization in animals 12 hours after injection of 1 mg tamoxifen (C, E, G). The CaMKCreERT2 transgene copy number was determined by Southern blot analysis using a probe that detects restriction fragments of different sizes for the endogenous CaMKIIα gene (upper band) and the CaMKCreERT2 transgene (lower band) (H, I, J).
Figure 2
Figure 2
CreERT2 fusion protein expression driven by the regulatory elements of the CaMKIIα gene allows targeting of specific regions in the adult brain upon tamoxifen treatment. (A, B) β-galactosidase staining of brain slices revealed that animals harboring a ROSA26 Cre reporter allele and the CaMKCreERT2 transgene (R26R/CaMKCreERT2, two copies) show low Cre activity in absence of the ligand only within the hippocampus (Hc) (A), but strong staining within the cortex (Co) and hippocampus (Hc) upon tamoxifen treatment (B). (D-F) Analysis of β-galactosidase positive neurons using cryosections of the hippocampus of R26R/CaMKCreERT2 mice revealed sparse recombination in the granular neurons of the dentate gyrus (DG) and in hippocampal neurons of the CA1 region in the absence of tamoxifen. (C) β-galactosidase staining of a brain slice isolated from an animal harboring a ROSA26 Cre reporter allele and the constitutive CaMKCre transgene (R26R/CaMKCre) revealed recombination in the hippocampus and cortex as well as recombination in olfactory bulbs (Ob), striatum (St) and cerebellum (Ce). (G-I) Recombination of a conditional GR allele achieved using the CaMKCreERT2 transgene (two copies) plus tamoxifen treatment (2 × 1 mg tamoxifen for five consecutive days) is comparable to recombination obtained using the constitutive CaMKCre transgene. Recombination levels detected in distinct brain regions (Co, Hc, Ht = hypothalamus, Ce) of mice heterozygous for the conditional GR allele (GRflox) and either the constitutive (GRflox/wt/CaMKCre) (I) or the inducible (GRflox/wt/CaMKCreERT2, tamoxifen-treated) (G) Cre transgene (two copies) were determined using Southern blot analysis. The Southern blot strategy is depicted in (H), black boxes represent exons three and four of the GR gene, the arrowheads the loxP sites and S marks SacI recognition sites. The probe detects in case of the GRflox and the GRwt allele a DNA fragment of similar size and in case of the GRnull allele a smaller DNA fragment. The bands were quantified by phosphoimager and the percentage of recombination for each region is indicated. Animals for Cre reporter and Southern blot analysis were treated with 1 mg tamoxifen or vehicle twice a day for five consecutive days and sacrificed ten days after the last injection.
Figure 3
Figure 3
Ablation of GR protein expression in neurons of the adult brain upon tamoxifen treatment. Vibratome-sections of mice homozygous for a conditional GR allele (GRflox) and heterozygous for the CaMKCreERT2 transgene (GRCaMKCreERT2 mice) have been analyzed by immunohistochemistry. GR protein is expressed in vehicle-treated GRCaMKCreERT2 mice (A, D, G, J, M), whereas loss of GR expression upon tamoxifen treatment is observed in brain regions (B, E, H, K, N) that display strong CreERT2 expression (C, F, I, L, O). The analyzed GRCaMKCreERT2 mice harbor two copies of the CaMKCreERT2 transgene. Depicted are brain regions that have been identified as target regions using ROSA26 reporter mice and/or show strong GR expression in controls: cortex (A-C), CA1 region of the hippocampus (D-F), dentate gyrus (G-I), PVN (J-L) as well as central (ceA) and basolateral amygdala (blA) (M-O). The animals were injected with 1 mg tamoxifen or vehicle twice a day for five consecutive days and sacrificed ten days after the last injection. In addition, Cre transgenic mice were injected with tamoxifen 12 hours before analysis to obtain a nuclear staining for the CreERT2 fusion protein.
Figure 4
Figure 4
CreERT2-mediated recombination within the adult hippocampus depends on the transgene copy number and the tamoxifen dose. Pyramidal neurons in the hippocampal CA1 region of GRCaMKCreERT2 and control animals were analyzed by immunohistochemistry on vibratome-sections using an antibody against GR. Mice were injected with tamoxifen for five consecutive days either once per day (5 mg) (A, C, E, G) or twice per day (10 mg) (B, D, F, H) and sacrificed ten days after the last injection. Treatment with 5 mg tamoxifen results in an incomplete loss of GR protein if one or two copies of the CaMKCreERT2 transgene are present (C, E), whereas four copies of the transgene are sufficient to induce complete GR protein loss (G). GRCaMKCreERT2 mice harboring two or four copies of the Cre transgene display complete loss of GR protein after injection of 10 mg tamoxifen (F, H). A visible but still incomplete loss of GR protein was seen in CaMKCreERT2 transgenic mice of the one-copy line treated with 10 mg tamoxifen (D) whereas tamoxifen-treated controls show no apparent loss of GR (A, B).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Anagnostopoulos AV, Mobraaten LE, Sharp JJ, Davisson MT. Transgenic and knockout databases: behavioral profiles of mouse mutants. Physiol Behav. 2001;73:675–689. doi: 10.1016/S0031-9384(01)00525-X. - DOI - PubMed
    1. Bolivar V, Cook M, Flaherty L. List of transgenic and knockout mice: behavioral profiles. Mamm Genome. 2000;11:260–274. doi: 10.1007/s003350010051. - DOI - PubMed
    1. Chen C, Tonegawa S. Molecular genetic analysis of synaptic plasticity, activity-dependent neural development, learning, and memory in the mammalian brain. Annu Rev Neurosci. 1997;20:157–184. doi: 10.1146/annurev.neuro.20.1.157. - DOI - PubMed
    1. Nagy A. Cre recombinase: the universal reagent for genome tailoring. Genesis. 2000;26:99–109. doi: 10.1002/(SICI)1526-968X(200002)26:2<99::AID-GENE1>3.0.CO;2-B. - DOI - PubMed
    1. Gu H, Marth JD, Orban PC, Mossmann H, Rajewsky K. Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science. 1994;265:103–106. doi: 10.1126/science.8016642. - DOI - PubMed

Publication types

MeSH terms

LinkOut - more resources