Post on 28-Jan-2016
Tools
• P element – a versatile transposon.
• P-element structure
Figure 14-16
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.
Transposon mutagenesis
• Many variants:– Simple– Enhancer traps– Fusion protein– Over/ectopic expression
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.
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.
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.
Screen fry- chromosomes molecularly to identify cases where imprecise excision lead to a deletion of part or all of fry.
Cis acting sequences GFP reporterPosition independent expression
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Assay of cis acting regulatory sequences
figure 5.16
Tool
• Collection of transposon insertions.
• Goal: an insertion in every gene.
Gal4
Gal80
UAS
No galactose – Gal4 does not activate transcription
Galactose
In presence of Galactose, Gal80 does not bind Gal4, GAL4 activates transcription
Gal4 enhancer trap insertions provide a wide range of cell type/tissue pattern/developmental stage specific gene expression drivers.
figure 5.17
A temperature sensitive GAL80 transgene is available that can be used to temporally control GAL4.
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.
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• Autonomous vs non-autonomous
Lineage Restrictions
Developmental Time
Log of clone frequency
Log of clone size
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.
Lineage restrictions
• How to get around the clone size vs clone frequency problem.
• Minute technique.
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.
Ways to generate clones that express a gene that neighboring
cells do not.• Flip out
P
a c t 5Cp ro m o te r
FRT FRT
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Po ly A Po ly A
G e ne o f inte re stP
Flip O ut