Identification of the genomic insertion site of Pmel-1 TCR α and β transgenes by next-generation sequencing.
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.
Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
Yun
Y
Ji
Advanced Biomedical Computing Center, Frederick National Laboratory for Cancer Research, Frederick, Maryland, United States of America.
Natalie
N
Abrams
Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
Wei
W
Zhu
Advanced Biomedical Computing Center, Frederick National Laboratory for Cancer Research, Frederick, Maryland, United States of America.
Eddie
E
Salinas
Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
Zhiya
Z
Yu
Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
Douglas C
DC
Palmer
Advanced Biomedical Computing Center, Frederick National Laboratory for Cancer Research, Frederick, Maryland, United States of America.
Parthav
P
Jailwala
Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
Zulmarie
Z
Franco
Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
Rahul
R
Roychoudhuri
Advanced Biomedical Computing Center, Frederick National Laboratory for Cancer Research, Frederick, Maryland, United States of America.
Eric
E
Stahlberg
Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
Luca
L
Gattinoni
Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
Nicholas P
NP
Restifo
D000818:Animals; D001483:Base Sequence; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology; D002874:Chromosome Mapping; D002889:Chromosomes, Human, Pair 2 / Q000737:chemistry*; D018628:Gene Dosage; D015870:Gene Expression; D005822:Genetic Vectors; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics* / Q000276:immunology; D019076:Transgenes; D058951:gp100 Melanoma Antigen / Q000235:genetics* / Q000276:immunology
D016693:Receptors, Antigen, T-Cell, alpha-beta; D058951:gp100 Melanoma Antigen