DNA Microscopy - Establishing an immuno protocol
Typ
Examensarbete för masterexamen
Master's Thesis
Master's Thesis
Program
Biotechnology (MPBIO), MSc
Publicerad
2024
Författare
Andersson, Anna
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
As we continue to explore and understand the world, the demands on our methods and techniques evolve too. DNA microscopy is a developing method that offers an alternative to conventional light microscopy. Instead of relying on light and a convex lens, it utilizes nature’s building blocks, DNA, to create spatial images and describe different proteins near one another using sequencing. Björn Högberg’s lab has previously shown the method to work in an artificial system and the project is now ready to start exploring one of the options for a cellular setup.
One possibility is to create a multi-omic system that utilizes antibodies to deliver target strands to specific cells, mimicking the original artificial setup. This requires the conjugation of designed DNA strands, targets, to the antibody. An appropriate protocol was developed and established in this thesis. The four cell lines used were A549, HeLa, MCF-7, and MDA-MB-231. In situ immunocytochemistry (ICC) was used to visually confirm the relative specificity of the four cell types to the nine different antibodies. Both SiteClick and oYo-Link were trialed for the conjugation of targets, using both ProFIRE and Amicon to purify the conjugates. The result showed that all four cell lines had general specificity to antibodies CD44 and BASP1, but only HeLa and A549 to SLC38A1. A549 was the only cell line that showed specificity for ALDH3A1 and AKR1B10. For the conjugation protocol SiteClick in combination with ProFIRE and Amicon gave the best result for conjugate concentration and purity.
The results of the thesis provide the beginning of a library with appropriate cell lines and antibodies to use in a cellular setup. It also establishes a protocol for the conjugation of target strands to antibodies. To fully realize a cellular version of the artificial system more study needs to be performed on appropriate cell lines and antibodies for a more complex setup. To further evaluate the conjugates’ effect on the DNA reactions the reaction requires sequencing and re-construction.
Beskrivning
Ämne/nyckelord
Microscopy , Immunocytochemistry , Conjugation , Spatial Transcriptomics