Pilot testing and validation of the utilized mouse models

6.3.1.1 Testing the activity of the Myf5^CreER driver in ERMS using the R26R^+/- reporter strain

In a pilot test, the activity of the Myf5^CreER driver was analyzed in ERMS of Ptch^del/+ mutant mice. This Cre driver was chosen, because human and murine ERMS from Ptchmutant mice highly express Myf5 ³²³. In order to ensure that the oncRas mutations indeed will be induced in ERMS of Ptch^del/+ mice, Ptch^del/+Myf5^CreER mice were bred to the cre-reporter strain R26R.

When the mice developed a palpable tumor, they were injected i.p. with tamoxifen or solvent for 5 consecutive days and were sacrificed 1 or 5 weeks thereafter. ERMS and skeletal muscle (SM) as well as brain, heart (negative controls, not shown) and intestine (positive control due to endogenous β-galactosidase activity, not shown) were isolated and subjected to X-Gal stainings (see section 5.4.3). Another positive control was SM from Rosa26-lacZ mice. These mice express lacZ and thus show high β-galactosidase activity in every organ of the body ³⁰⁶.

Tissue sections of SM and ERMS from solvent-treated Ptch^del/+Myf5^CreERR26R^+/- mice showed no X-Gal staining, whereas ERMS from tamoxifen-treated Ptch^del/+Myf5^CreERR26R^+/- mice stained positive (Fig. 41). The galactosidase activity visualized by X-Gal staining was detected in ERMS 1 or 5 weeks after treatment. Since no staining was detected in SM, it was concluded that the Myf5^CreER driver is primarily active in ERMS and also does not show any leakiness i.e. it is not active without tamoxifen. Nevertheless, the staining itself was not as strong as expected from literature ³⁰⁵.

Figure 41: X-Gal staining of skeletal muscle and ERMS isolated from solvent- or tamoxifen-treated Ptch^del/+Myf5^CreER/+R26R^+/- mice

Cryo-embedded sections of skeletal muscle (SM) and ERMS (RMS) from solvent- or tamoxifen-treated Ptch^del/+Myf5^CreER/+R26R^+/- mice were subjected to X-Gal staining. Cryo-embedded sections of SM from ROSA26-lacZ mice served as positive control. The arrows point to areas of staining. Pictures were taken at 600x fold magnification.

6.3.1.2 HRas, KRas and NRas are expressed in SM and ERMS of Ptch^del/+ mice

Next, the expression of HRas, KRas and NRas were analyzed by PCR and qRT-PCR in SM and ERMS from Ptch^del/+ mice. These analyses were done to confirm that the Ras isoforms are expressed mainly in ERMS: if Ras should not be expressed in ERMS from Ptch^del/+ mice, an activation of oncRas from the endogenous Ras locus is not possible. As shown in Fig. 42, HRas, KRas and NRas are expressed in SM and ERMS tissue, respectively (Fig. 42A, Fig. 42C and Fig. 42E, respectively). Moreover, qRT-PCR data showed that the level of HRas expression is equal in SM and ERMS, whereas KRas and NRas level are higher in ERMS compared to SM (Fig. 42B, Fig. 42D and Fig. 42F, respectively).

In addition, the Ras loci were sequenced to rule out endogenous mutations in the 3 genes.

However, all 3 Ras loci were wt in more than 5 analyzed RMS from Ptch^del/+ mice (data not shown).

Figure 42: Ras isoforms are expressed in SM and ERMS tissue samples of Ptch^del/+ mice

The basal expression of HRas (A, B), KRas (C, D) and NRas (E, F) were analyzed in skeletal muscle (SM) and ERMS (RMS) tissue samples of Ptch^del/+ mice by PCR (A, C, E) and quantified by qRT-PCR (B, D, E). The numbers in A, C and E indicate the mouse identification number. The expression in embryos at E12.5 served as positive control. The histograms shown in B, D and F represent qRT-PCR analyses and show the mean gene expression ± SEM of 7 tissue samples for SM and ERMS. Data were normalized to 18S rRNA (18S, left) or Tbp (right). For statistical analyses non-parametric t-tests (Mann Whitney) were performed. *p<0.05, **p<0.01,

***p<0.001 compared to expression level within SM.

6.3.1.3 Testing Myf5^CreER activity at the Ras loci in ERMS of Ptch^del/+oncRas^fl/+Myf5^CreER/+ mice

After having checked the activity of the Myf5^CreER driver in ERMS (see section 6.3.1.1), it was tested whether this Cre driver also efficiently targets the 3 oncogenic Ras loci. This was done because it is well known that the recombination efficiency of one and the same Cre driver can vary at different loci ^338-340. The recombination assays were performed on frozen or paraffin-embedded tissue samples from SM and ERMS of Ptch^del/+HRas^fl/+Myf5^CreER/+, Ptch^del/+KRas^fl/+Myf5^CreER/+ and Ptch^del/+NRas^fl/+Myf5^CreRE/+ mice (compare section 5.2.3.2).

Additionally, frozen tissue samples from these mice were used for a second round of genotyping to confirm the genetic setting (compare section 5.2.3.1).

The recombination assay for HRas was done on PCR-amplified cDNA by enzymatic digestion with BpmI. The enzyme recognizes the wt sequence (derived from the wt and the floxed locus), whereas the mutant HRas exon is not recognized due to the HRas^G12V mutation ³⁰³ (compare section 5.2.3.2.2). As already described, successful recombination was indicated by weak 209 bp and 93 bp digestion bands and a strong undigested fragment of 302 bp.

SM tissue samples from untreated, solvent-treated (not shown) and tamoxifen-treated Ptch^del/+HRas^fl/+Myf5^CreER/+ mice showed a weak band for undigested transcript and a strong band for the digested, wt transcripts. The same bands were also observed in ERMS samples from untreated and solvent-treated (not shown) Ptch^del/+HRas^fl/+Myf5^CreER/+ mice (Fig. 43).

This might indicate that the Myf5^CreER driver is leaky at the HRas loci. However, it could also mean that the BpmI-mediated digestion of the cDNA was incomplete. Nevertheless, the recombination efficiency at the HRas locus increased in ERMS samples of tamoxifen-treated mice (Fig. 43). Thus, a strong band for the undigested, recombined transcript and a weak band for the digested, wt transcripts were observed, which indicated efficient recombination at the HRas locus in ERMS.

Figure 43: Recombination assays for the floxed HRas loci

Skeletal muscle (SM) and ERMS (RMS) tissue samples were subjected to RNA isolation and subsequent cDNA synthesis. Fragments of the HRas gene were PCR-amplified and then digested using BpmI. Products were separated by agarose gel electrophoresis. A successful recombination is indicated by weak digestion bands (209 bp and 93 bp) and a strong band for the undigested fragment (302 bp). No recombination should be indicated by strong digestion bands (209 bp and 93 bp) and lack of the undigested band (302 bp). However, due to either incomplete digestion or leakiness of the Cre driver at the floxed HRas locus the undigested band was always present. For more details, see text.

The recombination assay for KRas was done on PCR-amplified gDNA samples. A band of 304 bp represents the recombined KRas allele, whereas a band of 270 bp represents the wt KRas allele. Due to the fact that all mice were heterozygous for the floxed Ras alleles, efficient recombination at the KRas locus was demonstrated by the occurrence of the 304 bp fragment in addition to the 270 bp band. A double band was clearly observed in ERMS samples from tamoxifen-treated mice, whereas it was only detected in very rare cases of untreated or solvent-treated (data not shown) mice (Fig. 44). This suggests that the Myf5^CreER driver might be leaky at the floxed KRas locus in very few cases. Due to these data the ERMS incidence of tamoxifen- and untreated Ptch^del/+KRas^fl/+Myf5^CreER/+ mice were compared, whereas all untreated Ptch^del/+KRas^fl/+Myf5^CreER/+ mice showing spontaneous recombination have been excluded from analyses (see section 6.3.2.3).

Figure 44: Recombination assays for the floxed KRas loci

Skeletal muscle (SM) and ERMS (RMS) tissue samples were subjected to gDNA isolation and subsequent PCR which was used to prove efficient recombination at the floxed KRas locus. Amplificates were separated by agarose gel electrophoresis and analyzed afterwards. A double band (270 bp for wt and 304 bp for the floxed KRas locus after recombination) indicated efficient recombination, whereas a single band (270 bp for wt KRas) indicated no recombination.

The recombination assay for NRas was also done with PCR-amplified gDNA samples. As already stated above, all mice were heterozygous for the floxed Ras alleles. Therefore, efficient recombination at the floxed NRas locus was demonstrated by the occurrence of the 521 bp fragment in addition to the 487 bp band that represents the wt NRas allele. The analysis revealed that the double band only occurred in ERMS samples isolated from tamoxifen-treated mice, whereas it was never seen in ERMS from untreated Ptch^del/+NRas^fl/+Myf5^CreER/+ mice (Fig. 45). However, in rare cases recombination also occurred in SM of tamoxifen-treated Ptch^del/+NRas^fl/+Myf5^CreER/+ mice (data not shown). Since the latter fact probably is of no importance for ERMS growth, all tamoxifen-treated Ptch^del/+NRas^fl/+Myf5^CreER/+ mice (and all untreated mice) with the correct genotype were included in the analysis described in section 6.3.2.4.

Figure 45: Recombination assays for the floxed NRas loci

Skeletal muscle (SM) and ERMS (RMS) tissue samples were subjected to gDNA isolation and subsequent PCR, which was used to prove efficient recombination at the floxed NRas locus. Amplificates were separated by agarose gel electrophoresis and analyzed afterwards. A double band (487 bp for wt and 521 bp for the recombined floxed NRas alleles) indicated efficient recombination, whereas a single band (487 bp for the wt NRas allele) indicated no recombination.

6.3.1.4 Testing Ras activity in ERMS of tamoxifen-treated Ptch^del/+oncRas^fl/+Myf5^CreER/+

mice

Next, the functionality of the expressed oncRas alleles was investigated. For this purpose, ERMS samples from tamoxifen-treated Ptch^del/+oncRas^fl/+Myf5^CreER/+ mice were analyzed in a Ras activity assay (experimental details are explained in section 5.3.6). The densitometrical analysis (Fig. 46A) of two independent experiments and a representative Western Blot (Fig. 46B) highlight an approximate twofold increase in Ras activity in tumor tissue of tamoxifen-treated Ptch^del/+ mice with oncRas mutation.

Figure 46: Ras is active in ERMS tissue samples of tamoxifen-treated Ptch^del/+oncRas^fl/+Myf5^CreER/+ mice Protein was isolated from tumor samples of tamoxifen-treated Ptch^del/+ and Ptch^del/+oncRas^fl/+Myf5^CreER/+

(oncHRas, oncKRas, oncNRas) mice. Afterwards cell lysates were subjected to a bead-based pull-down assay to precipitate active Ras. Whole lysates of the same samples were used to detect total Ras. Afterwards, precipitated and whole lysates were analyzed by Western Blot to detect the protein level of active Ras and total Ras with specific antibodies. Hsc70 served as loading control for total Ras. The relative Ras activity was calculated by normalization of active Ras to total Ras/Hsc70 and is displayed in histograms showing the mean Ras activity of 6 tumors each cohort ± SEM. Ras activity in ERMS from Ptch^del/+mice served as control and was set to 1 (A). A representative Western Blot is shown in (B). Protein names and sizes in kDa are displayed on the right side of the blot. The depicted results are representative for 6 tumors of each cohort analyzed in two independent experiments. For statistical analyses unpaired non-parametric t-tests (Mann Whitney) were performed. *p<0.05,

**p<0.01 compared to relative Ras activity within tumor tissue from Ptch^del/+ mice.

Taken together, the results described in section 6.3.1 show that ERMS of Ptch^del/+ mice express wt HRas, KRas and NRas. In order to activate oncRas expression in Ptch^del/+oncRas^fl/+ mice the Myf5^CreER driver was used. Indeed, tamoxifen-mediated activation of this driver induces recombination of the conditional floxed oncRas alleles primarily in the tumor tissue. Moreover, Ras activity assays verify the successful Ras activation in ERMS of tamoxifen-treated Ptch^del/+oncRas^fl/+Myf5^CreER/+ mice.