Where is cre expressed

Moreover, Cre transgenic mouse strains tend to display unspecific expression, and thus a knockout phenotype, in various cell types. The mechanisms causing these undesired effects are widely unknown. In the first place, the activity of many promoters in most cases is not fully understood. Hence, there might be developmental stages or other environmental factors affecting the activity of a promoter that have not yet been characterized.

When Cre expression occurs within an early embryonic stage, all cells derived from this lineage will carry the deleted gene. This may lead to unspecified genetic interactions, i. Thirdly, a number of articles on the effects of integration site on the expression pattern of a given transgene have been published. Termed position-effect variegation PEV , the phenomenon of mosaic expression has originally been described as the genetic cause of heterogeneously coloured eye-discs in mutants of Drosophila melanogaster [3] — [5].

Silencing is assumed to be due to site specific effects such as condensation of chromatin [6] , [7] , close proximity to the centromere [8] , transgene orientation and methylation induced silencing [9]. Furthermore, expression levels of a given transgene appears to correlate inversely with ageing [10]. These previous publications altogether suggest that various processes may cause impaired expression of transgenes.

We here demonstrated that this might, at least in some cases, precludes reliable prediction of tissue-specific gene disruption by tail-biopsy genotyping. By means of site directed recombination, mouse models with a liver specific inactivation of various proteins employing Cre expression under the control of the Albumin promoter Alb have been generated [11] — [14]. Specifically for the Alb promoter driven expression of Cre recombinase, it has been reported, that the maximum level of recombination in hepatocytes occurs at two weeks of age [15].

We have used this line to generate a liver-specific knock-out of the frataxin gene [13] , and have observed a unambiguously efficient rate of recombination in our initial study, i. Subsequently and to generate animals for several follow-up studies we observed inconsistencies between the phenotype initially observed [13] in comparison to animals that were derived from intercrosses of later generations carrying the Alb Cre construct.

Therefore we started to systematically dissect these apparent inconsistencies. We first analyzed the presence of randomly integrated Cre transgene by PCR which gives rise to a single fragment of bp based on previously described primers [16] derived from the original cDNA sequence of Cre recombinase Fig.

The presence of a loxP-flanked exon 4 of the frataxin gene [17] was confirmed by PCR as previously described [16] and gives rise to either a double band for heterozygotes Fig 1B , column 1 or a signal at bp for animals homozygous for the loxP site Fig.

Accordingly, panels A and B of Fig. Columns 0 and 1 depict negative and positive control, respectively also applies to subsequent panels B to E. A double band depicts heterozygous genotype column 1 , one band at bp only accordingly indicates homozygous loxP insertion columns 2 to 9. The shorter band stems from RNA with excised exon 4, the long band corresponds to the intact loxP flanked exon 4. To further evaluate the actual expression of the Cre transgene, we performed RNA expression analysis.

While omission of reverse transcription Fig. This indicates that despite the presence of a genomic Cre signal Fig. The imbalance presented by the misleading genomic Cre signal could be confirmed by RT-PCR against the targeted exon 4 of the gene under investigation.

The parental inheritance pattern may not matter for all Cre strains, but it is a good idea to compare results obtained from both maternal and paternal transmission whenever possible. Cre is often expressed from a randomly integrated transgene, but very few insertion sites are known. Random transgene integration can disrupt an endogenous mouse gene and cause unanticipated side effects.

Since hemizygous mice have a wildtype chromosome present, using hemizygous instead of homozygous Cre genotypes might minimize unintended consequences of random transgene insertion. Cre toxicity can also occur when expression is high enough to affect cell physiology , or when Cre recognizes genomic sequences that resemble loxP sites.

Cre can damage DNA in fibroblasts , gastric cells , and sperm in the absence of any floxed sequence. For these reasons, it is wise to include the Cre mouse itself without any floxed sequences as a control group in your experiments. The tamoxifen-inducible Cre strain, B6. Data collected by the JAX Cre Resource show that tamoxifen induction is more robust in certain tissues pancreas and smooth muscle, for example than others spleen and ovary.

Differences in recombination efficiency among tissue types were also noted in a similar strain, B6. Strains with maternal Cre expression in the oocyte can save time when converting floxed alleles to complete knockouts; no further breeding is needed to remove the Cre transgene after confirming that recombination took place. Other strains with similar characteristics include the Sox2-cre strains: , , and There is a Cre reporter, B6.

When Cre activity is especially high, however, the green fluorescence is silenced. This tool can be used to understand the relative intensity of Cre activity in different cells or tissues. The regression line is plotted and significance given. Not significant n. Our data indicated that the Foxp3 promoter is active or has been active in multipotent progenitor cells other than those leading directly to Treg ontogeny.

We reasoned that only cells with an active Foxp3 promoter would also be positive for YFP. This data further supports our contention that the Foxp3 promoter is only transiently active in multipotent progenitor cells that lead to neutrophils, B cells and CD8 T cells and other immune cells. Figure 3. All cells were stained with Zombie Aqua to discriminate live from dead cells.

Individual data points are shown along with means and SEMs. Moreover, this reporter mouse allows us to test for temporal stochastic activity of Foxp3 in a range of tissues. Tamoxifen readily crosses the placenta.

However, preliminary experiments showed that tamoxifen administrated to pregnant females prevented term birth. To circumvent this issue, we analyzed tdTomato expression in tissues of fetal mice.

Published expression data indicated that the Foxp3 gene may be active by embryonic day 12 E Foxp3 expression in the central nervous system was been reported on E Expression in the hemolymphoid system, particularly the developing thymus, as well as alimentary system by E Other groups however, have reported that Foxp3 activity at E This may speak either to the variable nature of Foxp3 expression or differences in analysis.

Given these premises, we opted to administer tamoxifen to two pregnant sibling females at E12 and E13 and then harvested lungs and spleens from E20 fetal mice and CLN from the does Figure 4A. Given that the half-life of Tamoxifen is 5—7 days, this would introduce a sufficient concentration into the systemic circulation to induce red fluorescent protein expression at E Foxp3 protein has not been detected in fetal mouse lungs at E Figure 4.

A Cartoon showing experimental timeline and design. Females Does that had evidence of a copulation plug on embryonic day 0 and that had gained weight by E12 compared to copulation plug minus siblings were orally administered tamoxifen on E12 and E Representative flow cytometry plots of CLN and a single fetal mouse spleen are shown. Numbers indicate the percentage of cells within a given gate.

This phenotype was not observed in our prior F 1 experimental mice. Note here that the Foxp3 transgene is X-linked. The appearance of this red coloration was striking and seemed widespread in non-immune tissue. In addition to skin cells, the red coloration was clearly visible in cells that comprise muscle, salivary glands and the capsule of CLN suggesting widespread expression of tdTomato in non-immune cells.

We reasoned that a cervical lymph node would give a clear picture of tdTomato expression in immune cells as well as non-immune cells that comprise the capsule, cortex and medullary stroma. These cells are presumably Th17 cells and were primarily located along the fringes of the B cell zone. Figure 5. Dissected CLN are shown in the lower panels of each montage. Red fluorescence was captured in the tdTomato channel.

Live mature and immature thymocytes were identified by flow cytometry. This analysis clearly showed that tdTomato was expressed in all four of the identified thymocyte populations. We decided to also look for tdTomato expression in these thymocyte populations.

Figure 6. Live mature and immature thymocytes were identified using Zombie Aqua, stained with a panel of rat anti-mouse mAbs and analyzed by flow cytometry. Numbers shown indicate the percentage of cells in a given gated population. Table 1. We developed fate-tracking reporter mouse strains to examine plasticity in Th17 and Treg cells in the context of a murine model of chronic periodontitis.

Both these cell types have been reported to undergo cytokine reprogramming during conditions of heightened chronic inflammation and a shifting cytokine milieu that develop during a number of CD4 T cell-mediated autoimmune diseases 6 , 7 , 13 , 19 , 50 , Translation of the tdTomato gene construct is tightly controlled in B6. Some CD8 T cells, however, have been reported to act as immuno-regulatory CD8 Treg cells expressing Foxp3 in a number of murine models of disease 44 — 48 , in bone homeostasis 52 and at low background levels It has been reported that CD8 Treg cells do not express CD28 or have low expression 46 , 53 and they appear to differentiate in the thymus of mice We chose the B6.

However, we found that tdTomato was activated in a broad range of CD4 T cells. This makes such a mouse model highly problematic for tracking authentic ex-Treg cells.

Cre expression during embryonic development. DAPI staining on coronal sections of embryos at E The regions analyzed in striatum and cortex by immunohistochemistry with Cre antibody are boxed. No Cre positive cells at E Cre expression appeared in the cortical plate, in the presumptive layer VI h-i.

Cre expression during postnatal development. Immunohistochemical localization of Cre. At P0 a-c and P3 d-f , striatal Cre is restricted to the patch compartment arrowheads. Expression in layer VI of the cortex is present already at birth. Transient and very weak expression is visible in the CA1 hippocampal region a,d,g and in layer IV of the cortex c,f,i. At P6 g-i , striatal Cre expression starts in the matrix compartment asterisk , between the patches arrowheads.

Expression in CA1 and cortex layer IV disappears progressively. In order to assess the specificity and the onset of Cre activity we have crossed D1Cre transgenic mice with the R26R reporter line [ 18 ] Figure 6. Interestingly, we were able to detect Cre activity in the tectum above the mesencephalic region Fig. The pattern of recombination induced by Cre recombinase was also analyzed in the adult brains Fig.

Cre activity in embryonic and adult brain in R26R D1Cre mice. The reporter mice are heterozygous for the R26R allele. Positive signal is present in the developing brain at this stage in the tectum above the mesencephalic fold boxed area , as shown also in d, arrowhead. In the adult brain, beta-galactosidase activity analyzed in coronal vibratome sections is present in striatum b,e , cortex b,f and hippocampus c.

We next examined whether Cre expression was confined to a specific subpopulation of striatal neurons. In the striatum, two subtypes of spiny neurons are involved in the so called "direct-" and "indirect-pathway". The direct pathway originates from striatal neurons projecting directly to the medial globus pallidus GPm and the substantia nigra pars reticulata SNr.

The neurons contributing to the indirect pathway are instead connected to these areas via the basal ganglia. Although it is still a matter of debate, it has been classically thought that the direct pathway neurons express the D1 dopamine receptor and the indirect-pathway neurons express the D2 receptor D2R [ 19 , 20 ]. In order to establish whether Cre expression is confined to a specific subpopulation, we retrogradely labeled neurons from the striatonigral pathway by stereotaxic injection of red fluorescent latex spheres into the substantia nigra pars reticulata and localized Cre within the striatum by immunofluorescence Fig.

Double labeling of neuron-specific class III beta-tubulin and Cre recombinase showed that the majority of striatal neurons express the Cre recombinase driven by the Drd1a gene Fig. This experiment showed that Cre expression was not restricted to the retrogradely labeled neurons which correspond to neurons of the direct pathway that project directly to the substantia nigra.

Cre expression in striatal neurons. Cre was labeled by immunofluorescence green fluorescence. To further assess the subpopulation specificity of the D1Cre transgene, we checked whether also D2R-positive cells express Cre protein in the striatum.

Therefore, we performed in situ hybridization by using a D2R-specific riboprobe followed by immunohistochemistry with Cre-specific antibody Fig. It has to be mentioned that some studies indicate that there is only very little overlap in the number of neurons expressing both receptors [ 19 , 21 ]. According to these studies we were not expecting expression of D1Cre in D2R positive cells to such an extent.

However, depending on the sensitivity and resolution of the adopted technical approach, others studies reveal that there is a higher degree of overlap [ 22 ] and that possibly all dopamine receptor-containing neurons, in the striatum, express both receptor types [ 23 , 24 ].

These results underscore the importance of verifying recombination in the context of the loxP modified allele of interest. Recombination is shown in striatum a-f , hippocampus g-l and cortex m-r.

Control animals are devoid of the D1Cre transgene while mutant animals carry the D1Cre trangene, as indicated. The hippocampus CA2 field is indicated by arrowheads. The cortical layers are indicated. The pattern of expression obtained with the D1Cre YAC transgene was very reproducible between transgenic lines.

The expression level was essentially only dependent on the copy number of the transgene. These observations illustrate well the properties of transgenes based on large DNA segments: position-independent and copy-number dependent expression of the transgene. This correlation between expression pattern and copy number usually does not apply to plasmid-based constructs [ 28 ].

The pattern of Cre expression as seen in the D1Cre transgenic animals is very close to the pattern of expression of the endogenous Drd1a gene reported in previous in situ hybridization studies [ 29 ].