The ATRX cDNA is prone to bacterial IS10 element insertions that alter its structure.
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.
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.
David
D
Valle-García
Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN USA.
Lyra M
LM
Griffiths
Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN USA ; Howard Hughes Medical Institute, Chevy Chase, MD USA.
Michael A
MA
Dyer
Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, 1470 Madison Avenue, New York, NY 10029 USA.
Emily
E
Bernstein
Instituto de Fisiología Celular, Departamento de Genética Molecular, Universidad Nacional Autónoma de México, Ciudad Universitaria, México DF, México.
Félix
F
Recillas-Targa
ATRX; ATRX over-expression; Chromatin remodeling; Cloning vector; IS10 element; Insertion element