A method for TAT-Cre recombinase-mediated floxed allele modification in ex vivo tissue slices

Author:

Cheong Sek-Shir1,Luis Tiago C.23,Stewart Michelle4,Hillier Rosie4,Hind Matthew15ORCID,Dean Charlotte H.1ORCID

Affiliation:

1. National Heart and Lung Institute (NHLI), Imperial College London 1 , London SW7 2AZ , UK

2. Centre for Inflammatory Diseases 2 , Department of Immunology and Inflammation , , London W12 0NN , UK

3. Imperial College London 2 , Department of Immunology and Inflammation , , London W12 0NN , UK

4. The Mary Lyon Centre at MRC Harwell 3 , Harwell Campus, Oxfordshire OX11 0RD , UK

5. National Institute for Health Research (NIHR) Respiratory Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust 4 , London SW3 6NP , UK

Abstract

ABSTRACT Precision-cut lung slices (PCLS) are used for a variety of applications. However, methods to manipulate genes in PCLS are currently limited. We developed a new method, TAT-Cre recombinase-mediated floxed allele modification in tissue slices (TReATS), to induce highly effective and temporally controlled gene deletion or activation in ex vivo PCLS. Treatment of PCLS from Rosa26-flox-stop-flox-EYFP mice with cell-permeant TAT-Cre recombinase induced ubiquitous EYFP protein expression, indicating successful Cre-mediated excision of the upstream loxP-flanked stop sequence. Quantitative real-time PCR confirmed induction of EYFP. We successfully replicated the TReATS method in PCLS from Vangl2flox/flox mice, leading to the deletion of loxP-flanked exon 4 of the Vangl2 gene. Cre-treated Vangl2flox/flox PCLS exhibited cytoskeletal abnormalities, a known phenotype caused by VANGL2 dysfunction. We report a new method that bypasses conventional Cre-Lox breeding, allowing rapid and highly effective gene manipulation in ex vivo tissue models.

Funder

Royal Brompton and Harefield NHS Foundation Trust

Mr and Mrs Youssef Mansour

Wellcome Trust

Royal Society

Imperial College London

Publisher

The Company of Biologists

Subject

General Biochemistry, Genetics and Molecular Biology,Immunology and Microbiology (miscellaneous),Medicine (miscellaneous),Neuroscience (miscellaneous)

Cited by 1 articles. 订阅此论文施引文献 订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献

1. First person – Sek-Shir Cheong;Disease Models & Mechanisms;2023-11-01

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3