1. The University of Manchester Faculty of Life Sciences Transposable elements BIOL20332/20972 GENETICS / Dev. Biol. RSM MODULE 2 Andreas Prokop

2. Making transgenic flies

5. Analysing gene expression wt - anti-slit

6. fly wt - anti-slit Analysing gene expression

7. wt - anti-slit Analysing gene expression

8. wt - anti-slit Analysing gene expression

9. wt - anti-slit slit-lacZ - anti-βGal Analysing gene expression

10. Important improvements of transposable elements that have shaped work with Drosophila

11. Difference: enhancer is not transgenic but endogenous Enhancer trap

12. P source of P-elements constitutively expressed non-excisable transposase source

13. Enhancer trap P

14. Rescue experiments

15. X-Gal4 ; UAS-lacZ A. Prokop

16. Experiments by group Groupslarval genotypestainingcomments 9 - 12, 29A-lacZ 2hr 37ºC, then o.N. 26ºC 5 groups 17 - 20B-lacZ2hr 37 ºC 4 groups 25 - 28, 31, 32C-lacZ2hr 26 ºC 6 groups 13 - 16D-lacZ 2hr 37ºC, then o.N. 26ºC 4 groups 21 - 24E-Gal4;UAS-lacZ2hr 37 ºC 4 groups 5 - 8F-Gal4;UAS-lacZ2hr 37 ºC 4 groups 1 - 4G-lacZ 2hr 37ºC, then o.N. 26ºC 4 groups

17. Performing enhancer trap screens A. Prokop

18. and identify the gene use the line as marker for experiments perform gain-of-function experiments A. Prokop Experimental applications for enhancer-trap lines

19. Carry out loss-of-function studies A. Prokop

20. Forward Genetics FORWARD GENETICS: identify genes that are responsible for a particular biological process or function (large-scale screens)

21. Reverse Genetics REVERSE GENETICS: unravel the functions behind specific genes of interest (using the fly as a "test tube") GOF approaches: gene is excessively or ectopically expressed or its function exaggerated (classical GOF alleles, targeted expression of genes - wild type alleles or constitutive active versions) LOF approaches: gene function is reduced (classical mutant alleles, RNA interference, expression of dominant-negative constructs, expression of single domain antibodies or anti-GFP nanobodies, genomic engineering - TALEN, CRISPR) Box 6: Awareness of potential genetic modifiers