Cloning and Mutagenesis of PTCHD1-GFP

The sequence of full‐length coding Ptchd1 mouse cDNA was amplified from the IMAGE cDNA clone 40095445 (GenBank accession number BC116312; Source BioSience) and cloned in pAcGFP1‐N vectors using In‐Fusion cloning strategy (Catalog no. 632501, Clontech) to generate PTCHD1‐GFP with GFP tag at the C‐terminal end (previously described in Ung et al. (2018 (link))). Importantly, the murine PTCHD1 protein shares the same amino acids length (888) with 98% of sequence identity between murine and human PTCHD1 proteins. The mutant PTCHD1‐GFP plasmids were generated using the Q5® Site‐Directed Mutagenesis Kit (New England Biolabs) according to the manufacturer's recommendations. The primer sequences for each mutagenesis are indicated in Table S1. The efficiency of the site‐directed mutagenesis was verified by Sanger Sequencing.

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Halewa J., Marouillat S., Dixneuf M., Thépault R., Ung D.C., Chatron N., Gérard B., Ghoumid J., Lesca G., Till M., Smol T., Couque N., Ruaud L., Chune V., Grotto S., Verloes A., Vuillaume M., Toutain A., Raynaud M, & Laumonnier F. (2021). Novel missense mutations in PTCHD1 alter its plasma membrane subcellular localization and cause intellectual disability and autism spectrum disorder. Human Mutation, 42(7), 848-861.

Publication 2021

In‐Fusion cloning strategy Q5® Site‐Directed Mutagenesis Kit

Amino acids Cdna Coding sequence Human ptchd1 proteins Mouse Murine Mutagenesis Plasmids Primer Protein Site directed mutagenesis Vectors

Corresponding Organization : Centre Hospitalier Universitaire de Tours

Other organizations : Centre National de la Recherche Scientifique, Université Claude Bernard Lyon 1, Hospices Civils de Lyon, Institut NeuroMyoGène, Hôpitaux Universitaires de Strasbourg, Université de Lille, Centre Hospitalier Universitaire de Lille, Hôpital Robert-Debré, Assistance Publique – Hôpitaux de Paris, Université Paris Cité, NeuroDiderot, Délégation Paris 7

Top 5 similar protocols

Variable analysis

independent variables

Mutagenesis of PTCHD1-GFP plasmid

dependent variables

Efficiency of site-directed mutagenesis

control variables

Sequence of full-length coding Ptchd1 mouse cDNA from the IMAGE cDNA clone 40095445
Cloning of PTCHD1-GFP with GFP tag at the C-terminal end
Amino acid length (888) and sequence identity (98%) between murine and human PTCHD1 proteins

controls

Positive control: None specified
Negative control: None specified

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