the university of manchester faculty of life sciences transposable elements biol20332/20972...

The

Uni

vers

ityof

Man

ches

ter

Facu

lty o

fLi

fe S

cien

ces Transposable

elements

BIOL20332/20972

GENETICS / Dev. Biol. RSM

MODULE 2

Andreas Prokop

Making transgenic flies

Making transgenic flies

Making transgenic flies

Analysing gene expression

wt - anti-slit

fly

wt - anti-slit

Analysing gene expression

wt - anti-slit

Analysing gene expression

wt - anti-slit

Analysing gene expression

wt - anti-slit

slit-lacZ - anti-βGal

Analysing gene expression

Important improvements of transposable elements that have

shaped work with Drosophila

Difference: enhancer is not transgenic but endogenous

Enhancer trap

Enhancer trap

Psource of

P-elementsconstitutively

expressednon-excisable

transposase source

Enhancer trap

P

Rescue experiments

X-Gal4 ; UAS-lacZ

A. P

roko

p

Experiments by group

Groups larval genotype staining comments

9 - 12, 29 A-lacZ 2hr 37ºC, then o.N. 26ºC 5 groups

17 - 20 B-lacZ 2hr 37ºC 4 groups

25 - 28, 31, 32 C-lacZ 2hr 26ºC 6 groups

13 - 16 D-lacZ 2hr 37ºC, then o.N. 26ºC 4 groups

21 - 24 E-Gal4;UAS-lacZ 2hr 37ºC 4 groups

5 - 8 F-Gal4;UAS-lacZ 2hr 37ºC 4 groups

1 - 4 G-lacZ 2hr 37ºC, then o.N. 26ºC 4 groups

Performing enhancer trap screens

A. P

roko

p

and identify the gene

use the line as marker for

experiments

perform gain-of-function

experiments

A. P

roko

p

Experimental applications for enhancer-trap lines

Carry out loss-of-function studies

A. P

roko

p

Forward Genetics

FORWARD GENETICS: identify genes that are

responsible for a particular biological process or function

(large-scale screens)

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