Engineered NFAT and AP-1 Transcription Factor Constructs

The pMIGR1 vector (Pear et al., 1998 (link)) was modified to encode either Thy1.1 or ZsGreen (Matz et al., 1999 (link)) downstream of the IRES site instead of GFP. Constructs encoding NFAT1, NFAT2, cJun, cFos, or their mutants were cloned into the multiple cloning site using the In-Fusion HD Cloning Kit (Clontech Laboratories). Vectors encoding NFAT1 (gift from Anjana Rao, ID 11791; Addgene), NFAT1-CA (gift from Anjana Rao, ID 11792; Addgene), NFAT2 (gift from Anjana Rao, ID 11101; Addgene; Monticelli and Rao, 2002 (link)), NFAT2-CA (gift from Anjana Rao, ID 11793; Addgene), cJun (gift from Axel Behrens, ID 47443; Addgene; Aguilera et al., 2011 (link)), and cFos (MC203181; Origene) were used as cDNA templates. Based on the sequence alignment of NFAT1 and NFAT2, the R471A/L472A/T538G point mutations, designed to mimic the R468A/I469A/T535G point mutations in NFAT1-CARIT that impair AP-1 binding (Macián et al., 2000 (link)), were introduced into NFAT2 and NFAT2-CA using PCR-directed site mutagenesis to create NFAT2-RIT and NFAT2-CARIT, respectively. TAM67, a dominant negative mutant of cJun lacking residues 3–122 (Brown et al., 1993 (link)), was cloned from the cJun vector.

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Seth A., Yokokura Y., Choi J.Y., Shyer J.A., Vidyarthi A, & Craft J. (2023). AP-1–independent NFAT signaling maintains follicular T cell function in infection and autoimmunity. The Journal of Experimental Medicine, 220(5), e20211110.

Publication 2023

In-Fusion HD Cloning Kit

Cdna Ires Mutagenesis Pear Point mutations Sequence alignment Vector

Corresponding Organization : Yale University

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Variable analysis

independent variables

Constructs encoding NFAT1, NFAT2, cJun, cFos, or their mutants

dependent variables

Not explicitly mentioned

control variables

PMIGR1 vector modified to encode either Thy1.1 or ZsGreen downstream of the IRES site instead of GFP

controls

Positive controls: Vectors encoding NFAT1, NFAT1-CA, NFAT2, NFAT2-CA, cJun, and cFos
Negative control: TAM67, a dominant negative mutant of cJun lacking residues 3–122

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