Download - Tools P element – a versatile transposon.. P-element structure Figure 14-16.

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Page 1: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

Tools

• P element – a versatile transposon.

Page 2: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

• P-element structure

Figure 14-16

Page 3: Tools P element – a versatile transposon.. P-element structure Figure 14-16.
Page 4: 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.

Page 5: Tools P element – a versatile transposon.. P-element structure Figure 14-16.
Page 6: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

Transposon mutagenesis

• Many variants:– Simple– Enhancer traps– Fusion protein– Over/ectopic expression

Page 7: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

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.

Page 8: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

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.

Page 9: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

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.

Page 10: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

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

Page 11: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

Cis acting sequences GFP reporterPosition independent expression

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Assay of cis acting regulatory sequences

Page 12: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

figure 5.16

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Page 14: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

Tool

• Collection of transposon insertions.

• Goal: an insertion in every gene.

Page 15: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

Gal4

Gal80

UAS

No galactose – Gal4 does not activate transcription

Galactose

In presence of Galactose, Gal80 does not bind Gal4, GAL4 activates transcription

Page 16: Tools P element – a versatile transposon.. P-element structure Figure 14-16.
Page 17: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

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

Page 18: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

figure 5.17

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Page 20: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

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

Page 21: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

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.

Page 22: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

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Page 24: Tools P element – a versatile transposon.. P-element structure Figure 14-16.
Page 25: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

• Autonomous vs non-autonomous

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Page 30: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

Lineage Restrictions

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Page 32: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

Developmental Time

Log of clone frequency

Log of clone size

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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.

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Page 35: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

Lineage restrictions

• How to get around the clone size vs clone frequency problem.

• Minute technique.

Page 36: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

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.

Page 37: Tools P element – a versatile transposon.. P-element structure Figure 14-16.
Page 38: Tools P element – a versatile transposon.. P-element structure Figure 14-16.
Page 39: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

Ways to generate clones that express a gene that neighboring

cells do not.• Flip out

Page 40: Tools P element – a versatile transposon.. P-element structure Figure 14-16.

P

a c t 5Cp ro m o te r

FRT FRT

y

Po ly A Po ly A

G e ne o f inte re stP

Flip O ut

Page 41: Tools P element – a versatile transposon.. P-element structure Figure 14-16.
Page 42: Tools P element – a versatile transposon.. P-element structure Figure 14-16.