To date, little is known about the physiological and pathophysiological roles of alkylglycerol monooxygenase (AGMO)1, an enzyme responsible for the degradation of ether lipids. Therefore, Sailer and her colleagues generated a novel AGMO knockout (KO) mouse model using EUCOMM stem cells that contain the Cre/LoxP system for investigation2.
Unexpected structural event fools routine genotyping
Since PCR genotyping yielded peculiar results, the researchers decided to perform detailed genomic analysis, using TLA and nanopore sequencing, to solve the conundrum2. TLA data confirmed that the integration occurred at the intended location on chromosome 12 and revealed that the genetic manipulation did not precipitate any additional integrations in other parts of the genome2. Using qPCR-based genotyping, TLA and nanopore sequencing, the researchers found that a 94 kb genomic segment of the AGMO wild-type gene had been duplicated at the locus of homologous recombination2. Nevertheless, because the engineered mouse was still deficient in AGMO enzyme activity, this provided sufficient grounds to continue using the generated model to further investigate the physiological influence of AGMO2.
The stumbling blocks of conventional techniques
The present study is one of the many examples that account how a genetic duplication can easily fool genotyping by routine PCR. Moreover, conventional assays such as FISH or long-range PCR were also unable to resolve this unexpected large structural variation. Therefore, these limitations underscore the pitfalls of classical routine genotyping strategies to profile structural variations in transgenic models3,4.
One-stop shop for the complete genetic analysis of transgenic models
To this end, TLA positions itself as an excellent candidate for the robust detection of any structural variants (including structural variations) in and around (trans)genes of interest. In fact, TLA generates deep nucleotide coverage and is used to:
- map transgene integration sites at base-pair resolution;
- accurately assess whether large structural variations may have accompanied recombination event;
- provide copy number estimation upon profiling transgenic animals.
Recognition of TLA for the improved QC of engineered animal models
In this paper, Sailer and her colleagues highlight that “nanopore sequencing and TLA now allow detecting and mapping more complex genome lesions in a cost-convenient matter”. With growing scientific evidence that homologous recombination events can induce unexpected large structural variations5, there is a clear need for more careful and standardized quality assessments, in terms of genomic validation, of transgenic animal models in the near future.With almost a decade of experience, many researchers around the world have been turning to our TLA technology for the improved QC of genetically engineered animal models. In fact, TLA has been mentioned in various publication by the likes of The Jackson Laboratory and Genentech. To learn more on how TLA can be of relevance to your work, we invite you to join our upcoming webinar entitled: “Complete and unbiased genetic characterization for the QC of transgenic animal models” which will be held on: Thursday, the 15th of April 2021 at 4PM CET/10AM ET.
Interested, but unable to attend? Sign up anyway and we will make sure to send you a direct link to the recorded session afterwards!
References
[1] Sailer S, Keller MA, Werner ER, Watschinger K. The emerging physiological role of AGMO 10 years after its gene identification. Life (Basel). 2021;11:2.
[2] Sailer S, Coassin S, Lackner K, Fischer C, McNeill E, Streiter G, Kremser C, Maglione M, Green CM, Moralli D, Moschen AR, Keller MA, Golderer G, Werner-Felmayer G, Tegeder I, Channon KM, Davies B, Werner ER, Watschinger K. When the genome bluffs: a tandem duplication event during generation of a novel Agmo knockout mouse model fools routine genotyping. Cell Biosci. 2021 Mar 16;11(1):54. doi: 10.1186/s13578-021-00566-9. PMID: 33726865; PMCID: PMC7962373.
[3] Chaisson MJP, Sanders AD, Zhao X, Malhotra A, Porubsky D, Rausch T, et al. Multi-platform discovery of haplotype-resolved structural variation in human genomes. Nat Commun. 2019;10(1):1784.
[4] Sedlazeck FJ, Rescheneder P, Smolka M, Fang H, Nattestad M, von Haeseler A, et al. Accurate detection of complex structural variations using single-molecule sequencing. Nat Methods. 2018;15(6):461–8.
[5] Guirouilh-Barbat J, Lambert S, Bertrand P, Lopez BS. Is homologous recombination really an error-free process? Front Genet. 2014;5:1.
