Valle-García David, Griffiths Lyra M, Dyer Michael A, Bernstein Emily, Recillas-Targa Félix
Instituto de Fisiología Celular, Departamento de Genética Molecular, Universidad Nacional Autónoma de México, Ciudad Universitaria, México DF, México ; Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, 1470 Madison Avenue, New York, NY 10029 USA.
Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN USA.
Springerplus. 2014 May 2;3:222. doi: 10.1186/2193-1801-3-222. eCollection 2014.
The SWI/SNF-like chromatin-remodeling protein ATRX has emerged as a key factor in the regulation of α-globin gene expression, incorporation of histone variants into the chromatin template and, more recently, as a frequently mutated gene across a wide spectrum of cancers. Therefore, the availability of a functional ATRX cDNA for expression studies is a valuable tool for the scientific community. We have identified two independent transposon insertions of a bacterial IS10 element into exon 8 of ATRX isoform 2 coding sequence in two different plasmids derived from a single source. We demonstrate that these insertion events are common and there is an insertion hotspot within the ATRX cDNA. Such IS10 insertions produce a truncated form of ATRX, which significantly compromises its nuclear localization. In turn, we describe ways to prevent IS10 insertion during propagation and cloning of ATRX-containing vectors, including optimal growth conditions, bacterial strains, and suggested sequencing strategies. Finally, we have generated an insertion-free plasmid that is available to the community for expression studies of ATRX.
SWI/SNF样染色质重塑蛋白ATRX已成为调控α-珠蛋白基因表达、组蛋白变体掺入染色质模板的关键因素，并且最近发现它是多种癌症中频繁发生突变的基因。因此，获得用于表达研究的功能性ATRX cDNA对科学界来说是一种有价值的工具。我们在源自单一来源的两种不同质粒中，鉴定出细菌IS10元件的两个独立转座子插入到ATRX同工型2编码序列的外显子8中。我们证明这些插入事件很常见，并且在ATRX cDNA内存在一个插入热点。这种IS10插入产生截短形式的ATRX，这严重损害了其核定位。相应地，我们描述了在含ATRX载体的传代和克隆过程中防止IS10插入的方法，包括最佳生长条件、细菌菌株和建议的测序策略。最后，我们构建了一种无插入的质粒，可供科学界用于ATRX的表达研究。
