1. Tools
P element – a versatile transposon.

2. P-element structure
Figure 14-16

4. Types of vectors General transformation. Reporter vectors.
Regulated expression vectors.
Gateway cloning system vectors.
FRT containing vectors.
φC31 site specific recombination vectors.
Fold back RNA vectors.

6. Transposon mutagenesis
Many variants:
Simple
Enhancer traps
Fusion protein
Over/ectopic expression

7. A screen using the P element as a mutagen.
w; P[w+lacW] X w;TMS, Δ23 Sb/Dr
w; P[w+lacW]/TMS, Δ23 Sb X fz th st in
Look for flies that are phenotypically fz or in.
These are likely to be due to a P insertion into fz or in.

8. P as a mutagen How to test if a mutation is due to a P insertion.
Can Δ23 induce reversion? Frequency usually 30-.1%.
Reversion is often imprecise – can lead to deletion.

9. Reversion of a P insertion (in this case P carries w+):
w; fryP/TM6 X TMS, 23, Sb/Dr
w; fryP/TMS, 23, Sb X w; TM6/DcxF
Screen for white eyed flies with normal bristles. These will be w; fryRV/TM6 (or w; fryRV/DcxF) .
Establish a stock that is w; fryRV/TM6 and test for fry function.

10. Screen fry- chromosomes molecularly to identify cases where imprecise excision lead to a deletion of part or all of fry.

11. Assay of cis acting regulatory sequences
Position independent expression
Cis acting sequences
GFP reporter + + + +  -

12. figure 5.16

14. Tool Collection of transposon insertions.
Goal: an insertion in every gene.

15. Gal4
Galactose
Gal80
UAS
No galactose – Gal4 does not activate transcription
In presence of Galactose, Gal80 does not bind Gal4, GAL4 activates transcription

17. Gal4 enhancer trap insertions provide a wide range of cell type/tissue pattern/developmental stage specific gene expression drivers.

18. figure 5.17

20. A temperature sensitive GAL80 transgene is available that can be used to temporally control GAL4.

21. Genetic Mosaics
Provides a cell marker that cannot be diluted out. Very valuable for tracing cell lineage.
Can use to study gene function.
Gets around some aspects of pleiotropy.
Allows additional functional tests of genes and pathways.

25. Autonomous vs non-autonomous

30. Lineage Restrictions

32. Log of clone size
Log of clone frequency
Developmental Time

33. Lineage Restrictions
Early attempts to identify lineage restrictions were hampered by the difficulty in obtaining large clones.
Data did show that clone shape was not random. For example, clones on the leg are long and thin (extended along the proximal distal axis.

35. Lineage restrictions
How to get around the clone size vs clone frequency problem.
Minute technique.

36. Lineage Restrictions Anterior - Posterior Compartment boundary.
Also a compartment for the expression of regulatory genes.
E.g. hedgehog and DPP are expressed along AP boundary.

39. Ways to generate clones that express a gene that neighboring cells do not.
Flip out