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Mcfarlane, Gus Rowan, 2016. Generating large-scale conditional knock-in mouse models using CRISPR/Cas9 in embryonic stem. Second cycle, A2E. Uppsala: SLU, Dept. of Animal Breeding and Genetics (until 231231)



The discovery and repurposing of CRISPR/Cas9 has revolutionized the field of genome editing and allowed for the development of mouse models with unprecedented simplicity and speed. The application of CRISPR/Cas9 in mouse embryonic stem cells represents a highly efficient and wellestablished method for generating large-scale conditional knock-in mouse models. This study employed a CRISPR/Cas9-mediated gene targeting approach in mouse embryonic stem cells to introduce a conditional knock-in construct within intron 11 of the Fbxw7 gene. Fbxw7 is mutated in a diverse range of human cancers, including colorectal cancer. As existing mutant Fbxw7 mouse models carry null alleles, the aim of this study was to create a mouse model carrying one of the commonly occurring point mutations in human cancers (Fbxw7(R482Q)). The construct integrated into the genome of mouse embryonic stem cells was a 5.5 kb Cre-dependent conditional knock-in that harboured the Fbxw7(R482Q) mutant allele and a cyan fluorescent protein reporter gene. Following screening and functional testing of the targeted embryonic stem cells, two confirmed clones were microinjected into wild type blastocysts. Subsequently, eight chimeric male mice were born from four litters. Two 95% male chimeras are currently breeding with wild type females in the hope of achieving germline transmission of the embryonic stem cell derived genes. Successful germline transmission will generate heterozygous Fbxw7(R482Q) mice. Once a breeding population is established the Fbxw7(R482Q) mice will be crossed with Villin-Cre mice to study the role Fbxw7(R482Q) in the development of colorectal cancer.

Main title:Generating large-scale conditional knock-in mouse models using CRISPR/Cas9 in embryonic stem
Authors:Mcfarlane, Gus Rowan
Supervisor:Andersson, Göran and Davies, Ben
Examiner:Åbrink, Magnus
Series:Examensarbete / SLU, Institutionen för husdjursgenetik
Volume/Sequential designation:511
Year of Publication:2016
Level and depth descriptor:Second cycle, A2E
Student's programme affiliation:Other
Supervising department:(VH) > Dept. of Animal Breeding and Genetics (until 231231)
Keywords:CRISPR/Cas9, conditional knock-in mice, transgenic mice, Fbxw7, colorectal cancer
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Subject. Use of subject categories until 2023-04-30.:Animal genetics and breeding
Human medicine, health, and safety
Deposited On:03 Oct 2016 12:20
Metadata Last Modified:03 Oct 2016 12:20

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