Results: 5

1.
Figure 4.

Figure 4. From: Alveolar rhabdomyosarcomas in conditional Pax3:Fkhr mice: cooperativity of Ink4a/ARF and Trp53 loss of function.

Alveolar rhabdomyosarcoma in Pax3:Fkhr Myf6-Cre mice with cell cycle control mutations. Three animals with Trp53 or Ink4a/ARF mutations that have developed classical alveolar rhabdomyosarcomas. (A,F,K) Gross appearance of the tumors of the tongue/jaw, chest/arm, and upper and lower extremities, respectively, marked with black arrowheads. (B,G,L) MicroCT of the tumor(s) for each animal demarcated by white arrows. (C,H,M) Appearance of the tumors (white arrowheads) at necropsy. (D,I,N) Hematoxylin and eosin staining of the tumors demonstrating classical alveolar histology and entrapped skeletal muscle (D, black arrowhead), strap cell (D, inset), and multinucleated rhabdomyoblasts (N, inset). (E,J,O) Immunohistochemistry for myogenin was positive for each tumor.

Charles Keller, et al. Genes Dev. 2004 November 1;18(21):2614-2626.
2.
Figure 5.

Figure 5. From: Alveolar rhabdomyosarcomas in conditional Pax3:Fkhr mice: cooperativity of Ink4a/ARF and Trp53 loss of function.

Representative immunohistochemical profile of alveolar rhabdomyosarcomas from Pax3P3Fa/wt Ink4a/ARFF2-3/WT Myf6ICNm/WT mice. Tumors expressed myogenic markers common to human alveolar rhabdomyosarcomas, including c-Met (A), Pax7 (B), and MyoD (C). (D) The cell cycle re-entry marker, Cyclin D1, was strongly expressed. (E) Other markers common to human and mouse alveolar rhabdomyosarcomas included C-MYC. (F) The anti-apoptotic protein, Bcl-XL, was strongly expressed and appeared to localize to the nuclear membrane as has been previously reported (Hsu et al. 1997). (Insets) Higher magnification of positively staining cells (white arrowhead).

Charles Keller, et al. Genes Dev. 2004 November 1;18(21):2614-2626.
3.
Figure 2.

Figure 2. From: Alveolar rhabdomyosarcomas in conditional Pax3:Fkhr mice: cooperativity of Ink4a/ARF and Trp53 loss of function.

Characterization of the Pax3:Fkhr, Myf6-Cre experimental system. (A) The Myf6-Cre allele demonstrates a skeletal-muscle-specific pattern of expression in an E15.5 fetus carrying the ROSA26 Cre-activatable LacZ marker. (B) Nuclear-localized Cre (green) and the nuclear satellite cell marker Pax7 (red) are not coexpressed in cross-sectioned skeletal muscle of Myf6-Cre mice. (C) Cre and the post-satellite cell myogenic transcription factor MyoD are coexpressed in skeletal muscle of Myf6-Cre mice. (D) When crossed to the Pax3:Fkhr conditional knock-in allele, the Myf6-Cre allele causes heterogeneous expression of the eYfp marker of Pax3:Fkhr activation in cross-sectioned skeletal muscle of a 6-week-old mouse (right), with little autofluorescence in tissue of a control mouse (left). (E) Genomic DNA PCR of the Pax3:Fkhr locus from a 6-week-old mouse carrying the Myf6-Cre allele confirms heterogeneous activation of Pax3:Fkhr within each skeletal muscle sample from varying epaxial and hypaxial sites. Activation of Pax3:Fkhr in testes is noted. (F) RT-PCR of skeletal muscle tissue from a 6-week-old mouse carrying both the Pax3:Fkhr knock-in allele and the Myf6-Cre allele demonstrates Pax3:Fkhr transcript expression for the tissues shown to undergo genomic rearrangement of the Pax3:Fkhr knock-in allele.

Charles Keller, et al. Genes Dev. 2004 November 1;18(21):2614-2626.
4.
Figure 3.

Figure 3. From: Alveolar rhabdomyosarcomas in conditional Pax3:Fkhr mice: cooperativity of Ink4a/ARF and Trp53 loss of function.

Alveolar rhabdomyosarcoma in Pax3:Fkhr Myf6-Cre mice. (A) A 12-month-old female Pax3P3Fa/WT Myf6ICNm/WT mouse was found to have a large mass (black arrowhead) arising from the left chest wall near the axilla. (B) Computed tomography scan revealed a well-circumscribed 13.8 × 6.4 × 9-mm mass (white arrowhead) arising from a more infiltrative base. A stellate area of calcification was indicative of central necrosis within the tumor. (C) Gross dissection revealed a highly vascular, rubbery tumor. (D) Low-power magnification of the hematoxylinand eosin-stained tumor (blue) demonstrates adherent skeletal muscle (pink). (E) Higher-power magnification of the hematoxylin- and eosin-stained tumor shows densely packed cells with scant cytoplasm, consistent with the solid variant of alveolar rhabdomyosarcoma. (F) A human solid variant of alveolar rhabdomyosarcoma is shown for comparison. (G) Immunohistochemistry for myogenin is positive. (H) Genotyping of two areas of the tumor reveals clonal conversion of the conditional P3Fm allele to an activated P3Fa allele.

Charles Keller, et al. Genes Dev. 2004 November 1;18(21):2614-2626.
5.
Figure 1.

Figure 1. From: Alveolar rhabdomyosarcomas in conditional Pax3:Fkhr mice: cooperativity of Ink4a/ARF and Trp53 loss of function.

Structure of targeted alleles. (A) For the Pax3:Fkhr-targeted knock-in allele, a targeting vector was designed for the insertion of a complex cassette into the 3′-region of the Pax3 gene, thereby modifying the last three of 10 exons. FRT sites flank the neomycin resistance gene (Neo) and allow excision of this gene upon breeding to an Flp-e mouse (Rodriguez et al. 2000). The P3Fp allele indicates the presence of the neomycin resistance gene (Neo+), whereas the P3Fm allele indicated the absence of the neomycin resistance gene (Neo-). LoxP sites flank exons 8-10 of Pax3 as well as an inserted polyadenylation/stop signal from the Complement2 (C2) gene. Prior to activation, the P3Fm allele will transcribe a full-length Pax3 transcript. Upon activation by CRE, exons 8-10 and the stop signal are removed and exon 7 of Pax3 is juxtaposed to exons 2 and 3 of Fkhr, thereby creating an activated Pax3:Fkhr fusion allele (P3Fa). An internal ribosomal entry site (IRES) downstream from Fkhr exon 3 allows for bicistronic expression of eYFP. The positions of PCR genotyping primers ck82, ck83, and ck10 are shown. (TK1) Thymidine kinase; (FRT) Flp-e recombinase recognition sequence; (RI) EcoRI restriction site; (RV) EcoRV restriction site; (UTR) untranslated region. (B) For the Fkhr conditional mouse line, a targeting vector was designed for which exons 2 and 3 were flanked by Lox511 sites. The F2-3p allele indicates the presence of the neomycin resistance gene (Neo+), whereas the F2-3m allele indicates the absence of the neomycin resistance gene (Neo-). In the presence of CRE, the inactivated DEL2-3 allele is generated. The positions of PCR genotyping primers ck142, ck143, and ck30 are shown. (C) For the Myf6-Cre allele, a targeting vector was designed for the insertion of a complex cassette into the 3′-region of the Myf6 gene. At an engineered XhoI site in the 3′-UTR, an IRES-Cre was inserted to allow bicistronic expression of Myf6 and Cre. An FRT-Neo-FRT cassette was inserted 3′ to IRES-Cre. The ICNp allele indicates the presence of the neomycin resistance gene (Neo+), whereas the ICNm allele indicates its absence. The positions of PCR genotyping primers ck219, ba88, and ba86 are shown.

Charles Keller, et al. Genes Dev. 2004 November 1;18(21):2614-2626.

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