Investigating The Role Of Tumour Suppressor Genes In Salamander Regeneration And Tumorigenesis Through CRISPR Screens

Popular Abstract

Looking At Tumour Suppressor Genes Of A Tumour Resistant Species

Salamanders like Pleurodeles waltl are tailed amphibians capable of regenerating complex structures like entire organs and limbs. Therefore, they are great models to study the process of regeneration. Regeneration involves cells of the injured tissue going through a lot of growth, division, and migration to reconstruct the original tissue. But these are properties often associated with tumours. Most animals protect themselves from tumours with the help of genes called tumour suppressors. If the expression of these genes are either reduced or affected, it can lead to cancers. A similar reduction of the expression of these genes are also seen in regeneration and therefore one would expect salamanders to be highly prone to tumours arising from faulty regenerative processes. But surprisingly, it is very rare and even attempts at using carcinogens (cancer inducing substances) to cause cancer do not work well in these animals. In the past, injecting carcinogens into the salamanders resulted in the formation of non-cancerous growths and some of the animals even formed extra limbs. This raises interesting questions as to why these animals are resistant to cancers? And if we can control cancer into regenerative outcomes?

To tackle these questions, we decided to look at how tumour suppressors might be involved in both these outcomes. We did this by using a revolutionary genome editing technique called CRISPR/Cas9 which is easy to design and use. This system has a Cas9 protein that cuts the genome at specific sites allowing us to delete genes that are of interest. In the study, we injected the Cas9 protein into salamander eggs using a tiny capillary glass needle. This allowed us to delete genes while the animal is still an egg. The animals that develop from these eggs will not carry the targeted gene and thereby allow us to study its function in the animal. Using this strategy we made different genetically modified animals that are termed as crispants.

Among the many tumour suppressors, we decided to target the retinoblastoma family of tumour suppressors (RB), responsible for causing childhood retinoblastoma tumour. After we cut out the RB genes, the RB crispants failed to feed and moved in unusual patterns when disturbed. Since they did not feed, the animals died a few weeks after hatching. Therefore, we decided to investigate if this was because of a tumour in their eyes by looking at thin slices of their eye tissue under a microscope. Fascinatingly, we did not see tumours but instead that their eyes had not developed properly. We also decided to test out if these crispants would regenerate normally. Therefore, we performed tail amputation experiments and saw that the crispants regenerated poorly. This suggests that there could be other factors that protect them from tumours or that they die before the tumour can develop. Since the tumour suppressor also affected regeneration, this could mean that cancer and regeneration share similar mechanisms. Research like this can tell us more about why these animals are resistant to cancers and why there is a preference for regeneration over cancer which could possibly allow us to cure cancer by transforming it into regenerative outcomes.

Master’s Project in Molecular Biology, Molecular Genetics and Biotechnology, 45 Credits 2022-2023, Department of Biology, Lund University

Supervisor: Nicholas Leigh, Division of Molecular Medicine and Gene Therapy, Lund University

• author: Sivaprasad, Rohit
• supervisor: Nicholas Leigh
• organization: Degree Projects in Molecular Biology
• course: MOBN02 20222
• year: 2023
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9121975
• date added to LUP: 2023-06-08 16:11:09
• date last changed: 2024-01-11 12:13:20