Creation and characterization of mice with a mutation disrupting binding of a transcriptional repressor of insulin-like growth factor 2

Kroscher, Kellie Ann

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https://hdl.handle.net/2142/98425 Copy

Description

Title

Creation and characterization of mice with a mutation disrupting binding of a transcriptional repressor of insulin-like growth factor 2

Author(s)

Kroscher, Kellie Ann

Issue Date

2017-07-20

Director of Research (if dissertation) or Advisor (if thesis)

Dilger, Anna C.

Department of Study

Animal Sciences

Discipline

Animal Sciences

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

M.S.

Degree Level

Thesis

Keyword(s)

• Insulin-like growth factor-2 (IGF2)
• Mice
• Transcription activator-like effector nucleases (TALEN)
• Single nucleotide polymorphism (SNP)

Abstract

Insulin-like growth factor-2 (IGF2) is a key regulator of myogenesis as it promotes differentiation of myoblasts during embryonic and fetal development. A single nucleotide polymorphism (SNP) identified in pigs in intron 3 of IGF2 disrupts a binding site for the transcriptional repressor ZBED6 leading to increased expression of IGF2. The SNP prevents binding of ZBED6 increasing IGF2 expression prenatally as well as postnatally compared with their wild type counterparts. Pigs with this mutation have a 10 % increase in muscle mass and a 15% decrease in subcutaneous fat deposition despite similar body weights. Though IGF2 is a highly conserved gene across species, similar mutations in IGF2 have not been detected. Therefore, the effect of mutations preventing the binding of ZBED6 to IGF2 in species other than pigs remains unknown. In order to characterize how a mutation in the ZBED6 binding site of IGF2 impacts growth, a mouse model was created using transcription activator-like effector nucleases (TALEN) with co-injection of oligo donors. Two founders were generated containing mutations that disrupted the ZBED6 binding site. One founder mouse contained the targeted G>A point mutation that mimicked the mutation in pigs, and the other founder had an A>G point mutation from the designed oligo donor on one allele and a G>A point mutation at the target site (A>G/G>A), with the other allele containing a C insertion in the target site (C+). All of these mutations inhibited the binding of ZBED6. Founders were bred to wild type (WT) females. The offspring produced contained either the C-insertion or A>G/G>A mutations and were bred to WT females to create two different mutant lines. Offspring lean and fat mass were evaluated through ECHO MRI at 21, 35, 49, 63, and 77 days of age. Offspring were euthanized at 21, 49, or 77 days of age and muscle, organ, and fat depot weights were collected. As expected, body weight, fat, and lean mass increased with increasing age (P<0.01). Mice with a mutation disrupting ZBED6 binding sites were 10.09-12.73 % heavier than their WT counterparts at 49d, 63d and 77d. Additionally, lean mass at 77d of age and fat mass at 45d and after were increased (P≤0.05) in mutated mice compared with WT mice as determined by ECHO MRI. However, as a percentage of body weight, lean and fat mass did not differ between genotypes (P≥0.12). The tibialis anterior was increased (P=0.03) by 20% in mutated mice compared with WT mice at 77d, but there were no differences in muscle fiber size and fiber cross sectional area (P≥0.64). Liver expression of IGF2 decreased with increasing age in all genotypes (P=0.02), and IGF2 expression was increased by 12-32 fold in mutated mice compared with their WT counterparts (P≤0.01). In contrast, protein quantification of IGF2 in serum of 77d mice did not differ between genotypes (P=0.16). We had hypothesized that mice with mutations disrupting the ZBED6 binding site in IGF2 would have increased muscle mass and reduced fat deposition at the same body weight, similar to the phenotype of mutant pigs. Therefore, the increased mass but apparent lack of altered muscle and fat deposition normalized to body weight in mutant mice was unexpected. These results suggest that disrupting ZBED6 binding in IGF2 affects species differently. It is possible that methylation patterns in tissues differ among species, affecting the way ZBED6 interacts with the binding site. This study provides insight into how IGF2 influences growth and muscle fiber development, and is the first to create a mutation in the ZBED6 binding site in mice.

Graduation Semester

2017-08

Type of Resource

text

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http://hdl.handle.net/2142/98425

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Copyright 2017 Kellie Kroscher

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