Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 3 de 3
Filter
Add more filters










Database
Language
Publication year range
1.
Nat Protoc ; 17(4): 1142-1188, 2022 04.
Article in English | MEDLINE | ID: mdl-35288718

ABSTRACT

Genetically engineered mouse models (GEMMs) transformed the study of organismal disease phenotypes but are limited by their lengthy generation in embryonic stem cells. Here, we describe methods for rapid and scalable genome engineering in somatic cells of the liver and pancreas through delivery of CRISPR components into living mice. We introduce the spectrum of genetic tools, delineate viral and nonviral CRISPR delivery strategies and describe a series of applications, ranging from gene editing and cancer modeling to chromosome engineering or CRISPR multiplexing and its spatio-temporal control. Beyond experimental design and execution, the protocol describes quantification of genetic and functional editing outcomes, including sequencing approaches, data analysis and interpretation. Compared to traditional knockout mice, somatic GEMMs face an increased risk for mouse-to-mouse variability because of the higher experimental demands of the procedures. The robust protocols described here will help unleash the full potential of somatic genome manipulation. Depending on the delivery method and envisaged application, the protocol takes 3-5 weeks.


Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Neoplasms , Animals , CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Gene Editing/methods , Liver , Mice , Mice, Knockout , Neoplasms/genetics , Pancreas
2.
Nat Commun ; 10(1): 1415, 2019 03 29.
Article in English | MEDLINE | ID: mdl-30926791

ABSTRACT

B-cell lymphoma (BCL) is the most common hematologic malignancy. While sequencing studies gave insights into BCL genetics, identification of non-mutated cancer genes remains challenging. Here, we describe PiggyBac transposon tools and mouse models for recessive screening and show their application to study clonal B-cell lymphomagenesis. In a genome-wide screen, we discover BCL genes related to diverse molecular processes, including signaling, transcriptional regulation, chromatin regulation, or RNA metabolism. Cross-species analyses show the efficiency of the screen to pinpoint human cancer drivers altered by non-genetic mechanisms, including clinically relevant genes dysregulated epigenetically, transcriptionally, or post-transcriptionally in human BCL. We also describe a CRISPR/Cas9-based in vivo platform for BCL functional genomics, and validate discovered genes, such as Rfx7, a transcription factor, and Phip, a chromatin regulator, which suppress lymphomagenesis in mice. Our study gives comprehensive insights into the molecular landscapes of BCL and underlines the power of genome-scale screening to inform biology.


Subject(s)
DNA Transposable Elements/genetics , Genetic Testing/methods , Lymphoma, B-Cell/genetics , Animals , CRISPR-Cas Systems/genetics , Clone Cells , Gene Dosage , Gene Expression Regulation, Neoplastic , Genes, Neoplasm , Genes, Tumor Suppressor , Genetic Association Studies , Humans , Loss of Heterozygosity , Lymphoma, B-Cell/pathology , Mice, Inbred C57BL , Mice, Transgenic , Receptors, Antigen, B-Cell/metabolism , Reproducibility of Results
3.
Nat Protoc ; 12(2): 289-309, 2017 Feb.
Article in English | MEDLINE | ID: mdl-28079877

ABSTRACT

Transposon-mediated forward genetics screening in mice has emerged as a powerful tool for cancer gene discovery. It pinpoints cancer drivers that are difficult to find with other approaches, thus complementing the sequencing-based census of human cancer genes. We describe here a large series of mouse lines for insertional mutagenesis that are compatible with two transposon systems, PiggyBac and Sleeping Beauty, and give guidance on the use of different engineered transposon variants for constitutive or tissue-specific cancer gene discovery screening. We also describe a method for semiquantitative transposon insertion site sequencing (QiSeq). The QiSeq library preparation protocol exploits acoustic DNA fragmentation to reduce bias inherent to widely used restriction-digestion-based approaches for ligation-mediated insertion site amplification. Extensive multiplexing in combination with next-generation sequencing allows affordable ultra-deep transposon insertion site recovery in high-throughput formats within 1 week. Finally, we describe principles of data analysis and interpretation for obtaining insights into cancer gene function and genetic tumor evolution.


Subject(s)
DNA Mutational Analysis/methods , DNA Transposable Elements/genetics , Genomics/methods , Mutagenesis, Insertional , Neoplasms/genetics , Animals , DNA Fragmentation , Gene Regulatory Networks , Humans , Mice , Models, Molecular , Mutagenesis , Nucleic Acid Conformation
SELECTION OF CITATIONS
SEARCH DETAIL
...