Masters Theses

Date of Award

8-2025

Degree Type

Thesis

Degree Name

Master of Science

Major

Animal Science

Major Professor

Jonathan E. Beever

Committee Members

Troy N. Rowan, Daniel J. Mathew

Abstract

The CRISPR/Cas9 system has been shown to improve specificity and efficacy of genome-engineering in livestock. There are two genes where molecular variation in different species has shown significant effects on increased lean growth and reduced adiposity: insulin-like growth factor 2 (IGF2) and myostatin (MSTN). In pigs, a G-to-A substitution in intron 3 of IGF2 (g.3072G>[greater than]A) disrupts a transcriptional repressor ZBED6 binding site and subsequently increases postnatal IGF2 expression. This is associated with increased lean muscle yield and intramuscular fat deposition while simultaneously decreasing backfat. In other livestock species, numerous MSTN loss-of-function (LOF) mutations have been reported. MSTN is a known early regulator of myoblast differentiation, and it’s LOF results in muscle hyperplasia and the commonly referred to “double-muscled” phenotype. Animals of this phenotype have significantly increased skeletal muscle mass, improved feed conversion, and decreased fat deposition. These mutations act synergistically as demonstrated by a mouse line possessing both IGF2 overexpression and MSTN LOF mutations. At 11 weeks of age, wild-type male and female mice exhibited average body weights of 28.54 ±[plus or minus] 0.60 g and 22.06 ± 0.44 g, respectively. Mice overexpressing Igf2 weighed 31.80 ± 0.60 g (males) and 25.24 ± 0.33 g (females), while Mstn LOF mice weighed 34.29 ± 0.65 g (males) and 27.82 ± 0.39 g (females). The combination mice showed the most pronounced increase in body mass, with average weights of 40.77 ± 1.08 g (males) and 33.17 ± 0.47 g (females). Based on these findings and the high degree of evolutionary conservation observed for these genes across species, we initiated CRISPR/Cas9-mediated genome editing of both loci in bovine fetal fibroblasts. This approach was undertaken with the expectation of eliciting comparable enhancements in somatic growth and skeletal muscle development. Numerous individual targeted modifications were confirmed through sequencing, and gRNA combinations are now being used to simultaneously target both loci. Successfully modified cell lines will be used for somatic cell nuclear transfer to produce live animals with germline modifications for subsequent progeny testing and assessment of the potential utility within commercial beef production systems.

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