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Revisiting the Promethean Dream: The Role of Activation‐induced Cytidine Deaminase in the Induction to Pluripotency
Author(s) -
Chakraborty Ritayan,
Chaudhuri Jayanta
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.1029.13
Subject(s) - cytidine deaminase , reprogramming , induced pluripotent stem cell , activation induced (cytidine) deaminase , sox2 , klf4 , germinal center , immunoglobulin class switching , biology , microbiology and biotechnology , embryonic stem cell , flow cytometry , somatic hypermutation , b cell , cell , chemistry , antibody , genetics , gene
Upon encountering antigens in the germinal centers of peripheral lymphoid organs, mature B‐lymphocytes undergo class switch recombination (CSR). CSR alters B cell expression between different immunoglobulin types. This mechanism requires activation induced cytidine deaminase (AID). However, recent speculation into AID activity has led to the conclusion that AID can function as an indirect demethylase. The aim of the experiment is the elucidation of AID's molecular mechanism during the development of induced pluripotent stem cells (iPSCs). We hypothesize that when a cocktail of 4 transcription factors Oct4, Klf4, Sox2 and cMyc are introduced into fully differentiated cells using a retroviral vector, the cell undergoes a reprogramming process that promotes its conversion to an iPSC. AID is thought to mediate this conversion. Using recombinant mice for AID mutations and chemically transfecting the retroviral cassette with Polybrene, AID expression was tested via Flow Cytometry and Western Blotting. Results with an LSRFortessa X‐20 Cell Analyzer and Zeiss Confocal lens microscope at 150x. The results indicated that upon infection with OKSM factors only AIDpositive cells began reprogramming showing certain early pluripotent factors and a down‐regulation of CSR while AID knockout cells failed to maintain the pluripotent state for an extended period of time.