Transgenesis Methods and Protocols

This detailed volume focuses on genotyping and validation in addition to information on how to produce gene edited cells and animals for research. Future advances in biomedical research will benefit greatly from the use of precise gene targeting of transgenes in the genome as CRISPR technology supersedes earlier methods that relied on random transgene integration, which this collection reflects. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, as well as tips for troubleshooting and avoiding known pitfalls. 

Transgenesis and Genome Engineering: A Historical Review.- Practical Application of the 3Rs in Rodent Transgenesis.- Genetic and Molecular Quality Control of Genetically Engineered Mice.- Genotyping Genome-Edited Founders and Subsequent Generation.- ASIS-Seq: Transgene Insertion Site Mapping by Nanopore Adaptive Sampling.- High Throughput Analysis of CRISPR-Cas9 Editing Outcomes in Cell and Animal Models Using CRIS.py.- Advanced Technologies and Automation in mES Cell Workflow.- Gene Editing in Mouse Zygotes Using the CRISPR/Cas9 System.- Floxing by Electroporating Single-Cell Embryos with Two CRISPR RNPs and Two ssODNs.- CRISPR/Cas9 Endonuclease Mediated Mouse Genome Editing of 1 Cell and/or 2 Cell Embryos by Electroporation, and the Use of Rad51 to Enhance Knock-In Allele Homozygosity via Inter Homolog Repair Mechanism.- Electroporation-Mediated CRISPR/Cas9 Genome Editing in Rat Zygotes.- CRISMERE Chromosome Engineering in Mouse and Rat.- Targeted Integration of Transgenesat the Mouse Gt(ROSA)26Sor Locus.- Improved Genome Editing via Oviductal Nucleic Acids Delivery (i-GONAD): Protocol Steps and Additional Notes.- Gene Targeting in Rat Embryonic Stem Cells.- Rat Embryonic Stem Cell Transgenesis.- CRISPR/Cas9-Mediated Genome Editing in Zebrafish.- Generation of Rabbit Chimeras by 8-Cell Stage Embryo Injection.- Genome Editing in Pigs.- Generation of Genome-Edited Chicken through Targeting of Primordial Germ Cells.