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Updated: Jun 22, 2026
A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging
Published on: July 14, 2016
Mice hypomorphic for Atr have increased DNA damage and abnormal checkpoint response
Ryan L Ragland1, Martin F Arlt, Elizabeth D Hughes
1Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109-0618, USA. ryanragland@yahoo.com
Abstract:
The ATR checkpoint pathway responds to DNA damage during the S/G2 phases of the cell cycle and is activated early in tumorigenesis. Investigation of ATR's role in development and tumorigenesis is complicated by the lethality of homozygous knockout mice and the limited effects of heterozygous deficiency. To overcome this limitation, we sought to create mice with a hypomorphic Atr mutation based on the ATR mutation in the human disease Seckel syndrome-1 (SCKL1). Homozygous SCKL1 mice were generated by targeted knock-in of the A --> G SCKL1 mutation. Western blot and RT-PCR analysis established that homozygotes have no reduction in Atr protein or increase in missplicing as is seen in humans. Thus, the A --> G substitution alone is not sufficient to reproduce in mice the effects that are seen in humans. However, homozygous SCKL1 mice that retain the neo cassette used for targeting have an estimated 66-82% reduction in total Atr protein levels due to missplicing into the neo cassette. Under conditions of APH-induced replication stress, primary fibroblasts from homozygous mice displayed an increase in overall chromosome damage and an increase in gaps and breaks at specific common fragile sites. In addition, mutant cells display a significant delay in checkpoint induction and an increase in DNA damage as assayed by Chk1 phosphorylation and gamma-H2ax levels, respectively. These mice provide a novel model system for studies of Atr deficiency and replication stress.
Insights
Researchers created a novel mouse model for Seckel syndrome-1 (SCKL1) by targeting the ATR gene. These mice exhibit reduced ATR protein levels, leading to increased DNA damage and replication stress, offering new insights into ATR deficiency.
Area of Science:
- Cell Biology
- Genetics
- Cancer Research
Background:
- The ATR (Ataxia Telangiectasia and Rad3-related) checkpoint pathway is crucial for responding to DNA damage during S/G2 phases.
- ATR's role in development and tumorigenesis is challenging to study due to homozygous lethality and limited heterozygous effects.
- A hypomorphic Atr mutation model was needed to overcome limitations in studying ATR deficiency.
Purpose of the Study:
- To create a mouse model with a hypomorphic Atr mutation, mimicking the Seckel syndrome-1 (SCKL1) human disease.
- To investigate the effects of reduced ATR protein levels on DNA damage and replication stress in mice.
Main Methods:
- Targeted knock-in of the SCKL1 A --> G mutation into the mouse Atr gene.
- Western blot and RT-PCR to assess Atr protein levels and splicing.
- Analysis of primary fibroblasts from homozygous mice under APH-induced replication stress.
Main Results:
- The A --> G substitution alone did not reduce Atr protein or increase missplicing in homozygotes.
- Homozygous SCKL1 mice retaining the neo cassette showed a 66-82% reduction in total Atr protein due to missplicing.
- Mutant fibroblasts exhibited increased chromosome damage, gaps, breaks at fragile sites, delayed checkpoint induction, and elevated DNA damage markers (Chk1 phosphorylation, gamma-H2ax).
Conclusions:
- The generated hypomorphic Atr mouse model effectively mimics aspects of ATR deficiency and replication stress.
- These mice serve as a valuable system for studying the consequences of ATR deficiency in a developmental and tumorigenesis context.
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