Cloning and Mutagenesis of Plasmid Constructs

Mouse Fzd6 cDNA was cloned into a Cherry-C1 plasmid (K4-Fzd6-Cherry) and pEGFP-C1 Mouse Ceslr1 cDNA that was cloned into a pEGFP-N1 plasmid (pEGFPN1-Celsr1), obtained from Dr. Elaine Fuchs’s laboratory (Devenport and Fuchs 2008 (link)) (The Rockefeller University, New York, USA). pcDNA3.1+/C-eGFP-VANGL1 construct (clone ID: OHu10988) was purchased from the Genscript Company (Piscataway, NJ 08854, USA). Fzd6 p.Gln88Glu and VANGL1 p.Gly644Val mutations were introduced into their wild-type (WT) constructs through site-directed mutagenesis using GeneArt® Site-Directed Mutagenesis System (Thermo Fisher Scientific, CAT#: A14604). All plasmids were validated by sequencing analyses. M50 Super 8 × TOPFlash (Veeman et al. 2003a (link), b (link)), human beta-catenin pcDNA3, and pcDNA3-S33Y beta-catenin (Kolligs et al. 1999 (link)) were purchased from Addgene (https://www.addgene.org/ ). The pAP1-Luc reporter construct (Part#: 219074) was purchased from Agilent.

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Tian T., Lei Y., Chen Y., Karki M., Jin L., Finnell R.H., Wang L, & Ren A. (2020). Somatic mutations in planar cell polarity genes in neural tissue from human fetuses with neural tube defects. Human genetics, 139(10), 1299-1314.

Publication 2020

GeneArt® Site-Directed Mutagenesis System M50 Super 8 × TOPFlash human beta-catenin pcDNA3 pcDNA3-S33Y beta-catenin

Beta catenin Cdna Celsr1 Cherry Clone Fzd6 Human Mouse Mutations Plasmids Sequencing analyses Site directed mutagenesis

Corresponding Organization :

Other organizations : Peking University, Baylor College of Medicine

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Variable analysis

independent variables

Fzd6 p.Gln88Glu mutation
VANGL1 p.Gly644Val mutation

dependent variables

Wnt signaling activity (measured by TOPFlash reporter)

control variables

Wild-type (WT) Fzd6 and VANGL1 constructs

positive controls

Human beta-catenin pcDNA3
PcDNA3-S33Y beta-catenin

negative controls

M50 Super 8 × TOPFlash
PAP1-Luc reporter construct

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