Identification of the genomic insertion site of Pmel-1 TCR α and ß transgenes by next-generation sequencing.
PLoS One
; 9(5): e96650, 2014.
Article
en En
| MEDLINE
| ID: mdl-24827921
ABSTRACT
The pmel-1 T cell receptor transgenic mouse has been extensively employed as an ideal model system to study the mechanisms of tumor immunology, CD8+ T cell differentiation, autoimmunity and adoptive immunotherapy. The 'zygosity' of the transgene affects the transgene expression levels and may compromise optimal breeding scheme design. However, the integration sites for the pmel-1 mouse have remained uncharacterized. This is also true for many other commonly used transgenic mice created before the modern era of rapid and inexpensive next-generation sequencing. Here, we show that whole genome sequencing can be used to determine the exact pmel-1 genomic integration site, even with relatively 'shallow' (8X) coverage. The results were used to develop a validated polymerase chain reaction-based genotyping assay. For the first time, we provide a quick and convenient polymerase chain reaction method to determine the dosage of pmel-1 transgene for this freely and publically available mouse resource. We also demonstrate that next-generation sequencing provides a feasible approach for mapping foreign DNA integration sites, even when information of the original vector sequences is only partially known.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Cromosomas Humanos Par 2
/
Mutagénesis Insercional
/
Receptores de Antígenos de Linfocitos T alfa-beta
/
Transgenes
/
Antígeno gp100 del Melanoma
Tipo de estudio:
Diagnostic_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
PLoS One
Asunto de la revista:
CIENCIA
/
MEDICINA
Año:
2014
Tipo del documento:
Article
País de afiliación:
Estados Unidos