Identification of reference genes for quantitative PCR during C3H10T1/2 chondrogenic differentiation

C3H10T1/2, a mouse mesenchymal stem cell line, is a well-known in vitro model of chondrogenesis that can be easily employed to recapitulate some of the mechanisms intervening in this process. Moreover, these cells can be used to validate the effect of candidate molecules identified by high throughput screening approaches applied to the development of targeted therapy for human disorders in which chondrogenic differentiation may be involved, as in conditions characterized by heterotopic endochondral bone formation. Chondrogenic differentiation of C3H10T1/2 cells can be monitored by applying quantitative polymerase chain reaction (qPCR), one of the most sensitive methods that allows detection of small dynamic changes in gene expression between samples obtained under different experimental conditions. In this work, we have used qPCR to monitor the expression of specific markers during chondrogenic differentiation of C3H10T1/2 cells in micromass cultures. Then we have applied the geNorm approach to identify the most stable reference genes suitable to get a robust normalization of the obtained expression data. Among 12 candidate reference genes (Ap3d1, Csnk2a2, Cdc40, Fbxw2, Fbxo38, Htatsf1, Mon2, Pak1ip1, Zfp91, 18S, ActB, GAPDH) we identified Mon2 and Ap3d1 as the most stable ones during chondrogenesis. ActB, GAPDH and 18S, the most commonly used in the literature, resulted to have an expression level too high compared to the differentiation markers (Sox9, Collagen type 2a1, Collagen type 10a1 and Collagen type 1a1), therefore are actually less recommended for these experimental conditions. In conclusion, we identified nine reference genes that can be equally used to obtain a robust normalization of the gene expression variation during the C3H10T1/2 chondrogenic differentiation

Transfection of the mutant MYH9 cDNA reproduces the most typical cellular phenotype of MYH9-related disease in different cell lines

ABSTRACT: BACKGROUND: Heterozygous mutations of MYH9, encoding the Non-Muscular Myosin Heavy Chain-IIA (NMMHC-IIA), cause a complex disorder named MYH9-related disease, characterized by a combination of different phenotypic features. At birth, patients present platelet macrocytosis, thrombocytopenia and leukocyte inclusions containing NMMHC-IIA. Moreover, later in life some of them develop the additional features of sensorineural hearing loss, cataracts and/or glomerulonephritis that sometimes leads to end stage renal failure. RESULTS: To clarify the mechanism by which the mutant NMMHC-IIA could cause phenotypic anomalies at the cellular level, we examined the effect of transfection of the full-length mutated D1424H MYH9 cDNAs. We have observed, by confocal microscopy, abnormal distribution of the protein and formation of rod-like aggregates reminiscent of the leukocyte inclusions found in patients. Co-transfection of differently labeled wild-type and mutant full-length cDNAs showed the simultaneous presence of both forms of the protein in the intracellular aggregates. CONCLUSION: These findings suggest that the NMMHC-IIA mutated in position 1424 is able to interact with the WT form in living cells, despite part of the mutant protein precipitates in non-functional aggregates. Transfection of the entire WT or mutant MYH9 in cell lines represents a powerful experimental model to investigate consequences of MYH9 mutations

High-throughput screening for modulators of ACVR1 transcription: discovery of potential therapeutics for fibrodysplasia ossificans progressiva.

open12noopenCappato, S; Tonachini, L; Giacopelli, F; Tirone, M; Galietta, Lj; Sormani, M; Giovenzana, A; Spinelli, Antonello E.; Canciani, B; Brunelli, S; Ravazzolo, R; Bocciardi, R.Cappato, S; Tonachini, L; Giacopelli, F; Tirone, M; Galietta, Lj; Sormani, M; Giovenzana, A; Spinelli, Antonello; Canciani, B; Brunelli, S; Ravazzolo, R; Bocciardi, R

Label-free, atomic force microscopy-based mapping of DNA intrinsic curvature for the nanoscale comparative analysis of bent duplexes

We propose a method for the characterization of the local intrinsic curvature of adsorbed DNA molecules. It relies on a novel statistical chain descriptor, namely the ensemble averaged product of curvatures for two nanosized segments, symmetrically placed on the contour of atomic force microscopy imaged chains. We demonstrate by theoretical arguments and experimental investigation of representative samples that the fine mapping of the average product along the molecular backbone generates a characteristic pattern of variation that effectively highlights all pairs of DNA tracts with large intrinsic curvature. The centrosymmetric character of the chain descriptor enables targetting strands with unknown orientation. This overcomes a remarkable limitation of the current experimental strategies that estimate curvature maps solely from the trajectories of end-labeled molecules or palindromes. As a consequence our approach paves the way for a reliable, unbiased, label-free comparative analysis of bent duplexes, aimed to detect local conformational changes of physical or biological relevance in large sample numbers. Notably, such an assay is virtually inaccessible to the automated intrinsic curvature computation algorithms proposed so far. We foresee several challenging applications, including the validation of DNA adsorption and bending models by experiments and the discrimination of specimens for genetic screening purposes

The Horizon of a Therapy for Rare Genetic Diseases: A “Druggable” Future for Fibrodysplasia Ossificans Progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic condition characterized by progressive extra-skeletal ossification leading to cumulative and severe disability. FOP has an extremely variable and episodic course and can be induced by trauma, infections, iatrogenic harms, immunization or can occur in an unpredictable way, without any recognizable trigger. The causative gene is ACVR1, encoding the Alk-2 type I receptor for bone morphogenetic proteins (BMPs). The signaling is initiated by BMP binding to a receptor complex consisting of type I and II molecules and can proceed into the cell through two main pathways, a canonical, SMAD-dependent signaling and a p38-mediated cascade. Most FOP patients carry the recurrent R206H substitution in the receptor Glycine-Serine rich (GS) domain, whereas a few other mutations are responsible for a limited number of cases. Mutations cause a dysregulation of the downstream BMP-dependent pathway and make mutated ACVR1 responsive to a non-canonical ligand, Activin A. There is no etiologic treatment for FOP. However, many efforts are currently ongoing to find specific therapies targeting the receptor activity and the downstream aberrant pathway at different levels or targeting cellular components and/or processes that are important in modifying the local environment leading to bone neo-formation

The role of the 3'UTR region in the regulation of the ACVR1/Alk-2 gene expression.

BACKGROUND: The ACVR1/Alk-2 gene, encoding a BMP type I receptor, is mutated in Fibrodysplasia Ossificans Progressiva, a severe form of heterotopic ossification. Regulation of ACVR1/Alk-2 expression, still poorly understood, is likely to be controlled by transcriptional and post-transcriptional mechanisms. In our work, we focused on the functional role of the 3'UTR region of the gene and on microRNAs as possible modulators of the ACVR1/Alk-2 expression. RESULTS: The ACVR1/Alk-2 3'UTR region consists of a 1.1 kb sequence harboring several putative, well-conserved binding sites for miRNAs in its proximal half, and AU-rich elements in the distal one, as assessed by bioinformatic analysis. The functional role of this region was tested in presence of transcription inhibitors and in transfection experiments in different cell lines, with a ACVR1/Alk-2-3'UTR reporter construct. By this transfection-based approach, we have also verified that three microRNAs, among those predicted to target ACVR1/Alk-2 gene by in silico analysis, can bind its 3'UTR sequence thereby modulating its expression. CONCLUSION: In this work we demonstrated that the ACVR1/Alk-2 transcript is unstable in presence of inhibitors of transcription. Functional analysis of the 3'UTR region by Luciferase reporter assays showed that it plays an inhibitory role on ACVR1/Alk-2 gene expression. Moreover, we found that specific miRNAs are involved in modulating ACVR1/Alk-2 gene expression as suggested by binding sites prediction in its 3'UTR sequence. In particular, we found that mir148b and mir365 were able to down-regulate ACVR1/Alk-2 expression, whereas mir26a showed a positive effect on its mRNA. Our data contribute to elucidate some of the mechanisms intervening in the modulation of ACVR1/Alk-2 expression. Considering that no specific and effective treatment of FOP is available, clarifying the basic mechanisms of the ACVR1/Alk-2 gene biology may provide means to develop innovative therapeutics approaches