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A Mouse Embryonic Stem Cell Bank for Inducible Overexpression of Human Chromosome 21 Genes

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A Mouse Embryonic Stem Cell Bank for Inducible Overexpression of Human Chromosome 21 Genes De Cegli et al. Genome Biology 2010, 11:R64 http://genomebiology.com/2010/11/6/R64 RESEARCH Open Access AResearch mouse embryonic stem cell bank for inducible overexpression of human chromosome 21 genes Rossella De Cegli†1, Antonio Romito†1,2, Simona Iacobacci1, Lei Mao3, Mario Lauria1, Anthony O Fedele1,4, Joachim Klose3, Christelle Borel5, Patrick Descombes6, Stylianos E Antonarakis5, Diego di Bernardo1, Sandro Banfi1, Andrea Ballabio1 and Gilda Cobellis*1,7 Abstract Background: Dosage imbalance is responsible for several genetic diseases, among which Down syndrome is caused by the trisomy of human chromosome 21. Results: To elucidate the extent to which the dosage imbalance of specific human chromosome 21 genes perturb distinct molecular pathways, we developed the first mouse embryonic stem (ES) cell bank of human chromosome 21 genes. The human chromosome 21-mouse ES cell bank includes, in triplicate clones, 32 human chromosome 21 genes, which can be overexpressed in an inducible manner. Each clone was transcriptionally profiled in inducing versus non- inducing conditions. Analysis of the transcriptional response yielded results that were consistent with the perturbed gene's known function. Comparison between mouse ES cells containing the whole human chromosome 21 (trisomic mouse ES cells) and mouse ES cells overexpressing single human chromosome 21 genes allowed us to evaluate the contribution of single genes to the trisomic mouse ES cell transcriptome. In addition, for the clones overexpressing the Runx1 gene, we compared the transcriptome changes with the corresponding protein changes by mass spectroscopy analysis. Conclusions: We determined that only a subset of genes produces a strong transcriptional response when overexpressed in mouse ES cells and that this effect can be predicted taking into account the basal gene expression level and the protein secondary structure. We showed that the human chromosome 21-mouse ES cell bank is an important resource, which may be instrumental towards a better understanding of Down syndrome and other human aneuploidy disorders. Background mining the molecular pathogenesis of aneuploidy disor- Aneuploidy refers to an abnormal copy number of ders [6]. genomic elements, and is one of the most common Our interest is focused on the elucidation of the molec- causes of morbidity and mortality in humans [1,2]. The ular basis of gene dosage imbalance in one of the most importance of aneuploidy is often neglected because clinically relevant and common forms of aneuploidy, most of its effects occur during embryonic and fetal Down syndrome (DS). DS, caused by the trisomy of development [3]. Initially, the term aneuploidy was human chromosome 21 (HSA21), is a complex condition restricted to the presence of supernumerary copies of characterized by several phenotypic features [6], some of whole chromosomes, or absence of chromosomes, but which are present in all patients while others occur only this definition has been extended to include deletions or in a fraction of affected individuals. In particular, cogni- duplications of sub-chromosomal regions [4,5]. Gene tive impairment, craniofacial dysmorphology and hypo- dosage imbalance represents the main factor in deter- tonia are the features present in all DS patients. On the other hand, congenital heart defects occur in only * Correspondence: [email protected] approximately 40% of patients. Moreover, duodenal 1 Telethon Institute of Genetics and Medicine, Via P. Castellino 111, Napoli, 80131, Italy stenosis/atresia, Hirschsprung disease and acute mega- † Contributed equally karyocytic leukemia occur 250-, 30- and 300-times more Full list of author information is available at the end of the article © 2010 De Cegli et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. De Cegli et al. Genome Biology 2010, 11:R64 Page 2 of 18 http://genomebiology.com/2010/11/6/R64 frequently, respectively, in patients with DS than in the onic stem (mES) cell clones capable of the inducible over- general population. Individuals with DS are affected by expression of each one of 32 selected genes, 29 murine these phenotypes to a variable extent, implying that many orthologs of HSA21 genes and 3 HSA21 coding phenotypic features of DS result from quantitative differ- sequences, under the control of the tetracycline-response ences in the expression of HSA21 genes. Understanding element (tetO). These genes include thirteen transcrip- the mechanisms by which the extra copy of HSA21 leads tion factors, one transcriptional activator, six protein to the complex and variable phenotypes observed in DS kinases and twelve proteins with diverse molecular func- patients [7,8] is a key challenge. tions. By transcriptome and proteome analysis, we deter- The DS phenotype is clearly the outcome of the extra mined that these clones, which are able to differentiate in copy of HSA21. However, this view does not completely different cell lineages, can be used to unveil the pathways address the mechanisms by which the phenotype arises. in which these genes are involved. We believe that this Korbel et al. [9] provided the highest resolution DS phe- resource represents a valuable tool to analyse the genetic notype map to date and identified distinct genomic pathways perturbed by the dosage imbalance of HSA21 regions that likely contribute to the manifestation of eight genes. DS features. Recent studies suggest that the effect of the elevated expression of particular HSA21 genes is respon- Results sible for specific aspects of the DS phenotype. Arron et al. Validation of an inducible/exchangeable system for [10] showed that some characteristics of the DS pheno- generation of transgenic mES cells type can be related to an increase in dosage expression of In order to generate a library of mES transgenic lines of two HSA21 genes, namely those encoding the transcrip- selected HSA21 genes, we used the ROSA-TET system. tional activator DSCR1-RCAN1 and the protein kinase This integrates the inducible expression of the Tet-off DYRK1A. These two proteins act synergistically to pre- system, the endogenous and ubiquitous expression from vent nuclear occupancy of nuclear factor of activated T the ROSA26 locus, and the convenience of transgene cells, namely cytoplasmic, calcineurin-dependent 1 exchange provided by the recombination-mediated cas- (NFATc) transcription factors, which are regulators of sette exchange (RMCE) system [19]. Briefly, coding vertebrate development. Recently, Baek et al. showed that sequences are cloned into an expression vector, driven by the increase in dosage of these two proteins is sufficient an inducible promoter (Tet-off), which can be easily inte- to confer significant suppression of tumour growth in grated into the ROSA26 locus through a cassette Ts65Dn mice [11], and that such resistance is a conse- exchange reaction. quence of a deficit in tumour angiogenesis arising from Understanding the expression kinetics of the system suppression of the calcineurin pathway [12]. Overexpres- was essential to standardizing the generation of the mES sion of a number of HSA21 genes, including Dyrk1a, library encoding the HSA21 genes. Towards this goal, we Synj1 and Sim2, results in learning and memory defects first tested the system by introducing the luciferase (Luc) in mouse models, suggesting that trisomy of these genes gene, cloned into an exchange vector. This enabled accu- may contribute to learning disability in DS patients [13- rate quantification of cassette exchange and gene induc- 15]. ibility, at both the RNA and protein level. To this end, we Many phenotypic features of DS are determined very prepared an exchange vector (pPTHC-Luc), which was early in development, when the tissue specification is not introduced into the EBRTcH3 ES cell line (EB3), carrying completely established [3]. Early postnatal development a yellow fluorescent protein (YFP) gene integrated in the of both human patients and DS mouse models showed ROSA26 locus. After the RMCE procedure, positive the reduced capability of neuronal precursor cells to cor- exchanged clones were identified by PCR (Additional file rectly generate fully differentiated neurons [16], contrib- 1a) and their inducibility verified using both reporter uting to the specific cognitive and developmental deficits genes. Quantitative PCR (q-PCR) analysis of Luc expres- seen in individuals with DS [17]. Canzonetta et al. [18] sion showed that the system was activated upon the showed that DYRK1A-REST perturbation has the poten- removal of Tetracycline (Tc) from the medium. In the tial to significantly contribute to the development of presence of Tc (0 hours; see Materials and methods), Luc defects in neuron number and altered morphology in DS. mRNA was undetectable, indicating that the background The premature reduction in REST levels could skew cell- expression level was almost zero, whereas a strong signal fate decisions to give rise to a relative depletion in the was detected 15 hours after Tc withdrawal, and still sus- number of neuronal progenitors. tained over a time window of 48 hours (Additional file The exact nature of these events and the role played by 1b). We then compared the mRNA level with the enzy- increased dosage of individual HSA21 genes remain matic activity of the protein Luc. To this end, we prepared unknown. To contribute to answering these questions, we the protein extracts of the Luc-inducible mES clones at have established a cell bank consisting of mouse embry- the same time points to quantify luminescence. In agree- De Cegli et al. Genome Biology 2010, 11:R64 Page 3 of 18 http://genomebiology.com/2010/11/6/R64 ment with the mRNA data, the enzymatic activity was It is therefore suitable for systematic expression of HSA21 undetectable in the presence of Tc, whereas a strong sig- cDNAs. nal was measurable 15 hours after Tc withdrawal, indicat- ing a correct induction of Luc translation (Additional file Cell bank: the HSA21 gene collection in mES cells 1b).
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