Using the GEMM-ESC strategy to study gene function in mouse models.
Preclinical in vivo validation of target genes for therapeutic intervention requires careful selection and characterization of the most suitable animal model in order to assess the role of these genes in a particular process or disease. To this end, genetically engineered mouse models (GEMMs) are typically used. However, the appropriate engineering of these models is often cumbersome and time consuming. Recently, we and others described a modular approach for fast-track modification of existing GEMMs by re-derivation of embryonic stem cells (ESCs) that can be modified by recombinase-mediated transgene insertion and subsequently used for the production of chimeric mice. This 'GEMM-ESC strategy' allows for rapid in vivo analysis of gene function in the chimeras and their offspring. Moreover, this strategy is compatible with CRISPR/Cas9-mediated genome editing. This protocol describes when and how to use the GEMM-ESC strategy effectively, and it provides a detailed procedure for re-deriving and manipulating GEMM-ESCs under feeder- and serum-free conditions. This strategy produces transgenic mice with the desired complex genotype faster than traditional methods: generation of validated GEMM-ESC clones for controlled transgene integration takes 9-12 months, and recombinase-mediated transgene integration and chimeric cohort production takes 2-3 months. The protocol requires skills in embryology, stem cell biology and molecular biology, and it is ideally performed within, or in close collaboration with, a transgenic facility.
Mouse Clinic for Cancer and Aging research (MCCA) Transgenic Core Facility, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Ivo J
IJ
Huijbers
Mouse Clinic for Cancer and Aging research (MCCA) Transgenic Core Facility, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Jessica
J
Del Bravo
Mouse Clinic for Cancer and Aging research (MCCA) Transgenic Core Facility, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Rahmen
R
Bin Ali
Mouse Clinic for Cancer and Aging research (MCCA) Transgenic Core Facility, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Colin
C
Pritchard
Mouse Clinic for Cancer and Aging research (MCCA) Transgenic Core Facility, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Tanya M
TM
Braumuller
Division of Molecular Pathology and Cancer Genomics Centre Netherlands, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Martine H
MH
van Miltenburg
Division of Molecular Pathology and Cancer Genomics Centre Netherlands, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Linda
L
Henneman
Division of Molecular Pathology and Cancer Genomics Centre Netherlands, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Ewa M
EM
Michalak
Division of Molecular Pathology and Cancer Genomics Centre Netherlands, The Netherlands Cancer Institute, Amsterdam, The Netherlands.|Division of Molecular Genetics, The Netherlands Cancer Institute, Amsterdam, The Netherlands.|Skoltech Center for Stem Cell Research, Moscow Region, Russia.
Anton
A
Berns
Division of Molecular Pathology and Cancer Genomics Centre Netherlands, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Jos
J
Jonkers
D000818:Animals; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D053595:Embryonic Stem Cells / Q000502:physiology*; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D008822:Mice, Transgenic; D011506:Proteins / Q000378:metabolism*; D011995:Recombination, Genetic; D019076:Transgenes
D011506:Proteins