Generation and Characterization of WIPI and ATG2A Constructs

DNA fragments encoding WIPI1 (NM_017983.7), WIPI2 (NM_016003.4), WIPI3 (NM_019613.4), WIPI4 (NM_001029896.2), and their variants were inserted into the retroviral plasmids pMRXIP (68 (link)), pMRXIB and pMRXIH together with 3 × FLAG tag or HaloTag7 (N2701, Promega, Madison, WI). DNA fragments encoding ATG2A (NM_015104.3) and its variants were inserted into pMRXIP (68 (link)) together with monomeric ultrastable green fluorescent protein (muGFP). DNA fragments encoding ATG2A (NM_015104.3) and its variants were also inserted into pMRXNo. The pMRXIB, pMRXIH and pMRXNo plasmids were constructed by replacing a puromycin-resistant gene cassette with a blasticidin-resistant gene cassette, a hygromycin B-resistant gene cassette, and an internal ribosomal entry site driven SNAP-tag (N9181S, New England BioLabs, Ipswich, MA), respectively. Mutated or truncated constructs were generated by PCR-mediated site-directed mutagenesis. The pMRXIP-GFP-LC3-RFP, pMRXIB-Halo-mGFP and pMRXNo-Halo-ratLC3B plasmids were constructed as described previously (41 (link),42 (link)).
For the generation of KO cell lines, guide RNAs (gRNAs) were cloned into pSpCas9(BB)-2A-GFP (PX458; a gift from Dr F. Zhang, Broad Institute of Massachusetts Institute of Technology; #48138, Addgene, Watertown, MA).

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Shimizu T., Tamura N., Nishimura T., Saito C., Yamamoto H, & Mizushima N. (2023). Comprehensive analysis of autophagic functions of WIPI family proteins and their implications for the pathogenesis of β-propeller associated neurodegeneration. Human Molecular Genetics, 32(16), 2623-2637.

Publication 2023

HaloTag7 pSpCas9(BB)-2A-GFP

A gene Cell lines Gene Hygromycin b Internal ribosomal entry site Plasmids Promega Puromycin Retroviral Rnas Site directed mutagenesis

Corresponding Organization :

Other organizations : The University of Tokyo, Japan Science and Technology Agency, Nippon Medical School

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Variable analysis

independent variables

DNA fragments encoding WIPI1 (NM_017983.7), WIPI2 (NM_016003.4), WIPI3 (NM_019613.4), WIPI4 (NM_001029896.2), and their variants
DNA fragments encoding ATG2A (NM_015104.3) and its variants
Mutated or truncated constructs generated by PCR-mediated site-directed mutagenesis

dependent variables

Measured outcomes (not explicitly mentioned)

control variables

3 × FLAG tag or HaloTag7
Monomeric ultrastable green fluorescent protein (muGFP)
SNAP-tag

positive controls

PMRXIP-GFP-LC3-RFP
PMRXIB-Halo-mGFP
PMRXNo-Halo-ratLC3B

negative controls

Not explicitly mentioned

Annotations

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