Characterizing Somatic Hypermutation and Gene Conversion in the Chicken DT40 Cell System

The selection of a suitable reference gene assures a reliable gene expression analysis when using the qPCR method. Normalization of the reaction is based on the basic metabolism genes. These genes show a constant, unregulated expression in all cells and function throughout their lifetime. In the current study, seven reference gene candidates were screened using RT-qPCR, to determine the best-matched pair of reference genes in the chicken DT40 cell line. The DT40 was derived from bursal lymphoma cells that were subjected to RAV-1 bird retroviral infection. It is a simplified in vitro model that allows tracking the direct interaction of stimulants on the lymphoid population and profiling of the hepatocellular B cell transcriptome. The reference gene analysis was carried out using statistical tools integrating four independent methods—geNorm, Best Keeper, NormFinder, delta Ct and RefFinder. Based on the selected reference genes, the relative gene expression analysis was done using the ddCt method. Complete relative gene expression study on a panel of the target genes revealed that proper selection of reference genes depending on the tissue eliminate decreases in data quality. The SDHA and RPL4 genes constitute stable internal controls as reference genes when analyzing gene expression in the DT40 cell line.

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Bcl6 Is Required for Somatic Hypermutation and Gene Conversion in Chicken DT40 Cells

PLoS ONE ◽

10.1371/journal.pone.0149146 ◽

2016 ◽

Vol 11 (2) ◽

pp. e0149146 ◽

Cited By ~ 5

Author(s):

Alan M. Williams ◽

Yaakov Maman ◽

Jukka Alinikula ◽

David G. Schatz

Keyword(s):

Gene Conversion ◽

Somatic Hypermutation ◽

Dt40 Cells

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CENP-O Class Proteins Form a Stable Complex and Are Required for Proper Kinetochore Function

Molecular Biology of the Cell ◽

10.1091/mbc.e07-06-0556 ◽

2008 ◽

Vol 19 (3) ◽

pp. 843-854 ◽

Cited By ~ 84

Author(s):

Tetsuya Hori ◽

Masahiro Okada ◽

Katsumi Maenaka ◽

Tatsuo Fukagawa

Keyword(s):

Sister Chromatid ◽

Proteasome Inhibitor ◽

Stable Complex ◽

Proteasome Inhibitor Mg132 ◽

Dt40 Cell ◽

Phenotype Analysis ◽

Functional Classes ◽

Sister Chromatid Separation ◽

Chicken Dt40 Cell ◽

Kinetochore Function

We previously identified a multisubunit complex (CENP-H/I complex) in kinetochores from human and chicken cells. We showed that the CENP-H/I complex is divided into three functional classes. In the present study, we investigated CENP-O class proteins, which include CENP-O, -P, -Q, -R, and -50 (U). We created chicken DT40 cell knockouts of each of these proteins, and we found that all knockout lines were viable, but that they showed slow proliferation and mitotic defects. Kinetochore localization of CENP-O, -P, -Q, and -50 was interdependent, but kinetochore localization of these proteins was observed in CENP-R–deficient cells. A coexpression assay in bacteria showed that CENP-O, -P, -Q, and -50 proteins form a stable complex that can associate with CENP-R. Phenotype analysis of knockout cells showed that all proteins except for CENP-R were required for recovery from spindle damage, and phosphorylation of CENP-50 was essential for recovery from spindle damage. We also found that treatment with the proteasome inhibitor MG132 partially rescued the severe mitotic phenotype observed in response to release from nocodazole block in CENP-50–deficient cells. This suggests that CENP-O class proteins are involved in the prevention of premature sister chromatid separation during recovery from spindle damage.

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