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The University
of Manchester
BIOL20332/20972
GENETICS / Dev. Biol.
RSM
Faculty of
Life Sciences
MODULE 2
Transposable
elements
Andreas Prokop
Making transgenic flies
Making transgenic flies
Making transgenic flies
Analysing gene expression
wt - anti-slit
Analysing gene expression
wt - anti-slit
fly
Analysing gene expression
wt - anti-slit
Analysing gene expression
wt - anti-slit
Analysing gene expression
wt - anti-slit
slit-lacZ - anti-βGal
Important improvements of
transposable elements that have
shaped work with Drosophila
Enhancer trap
P
Enhancer trap
P
Enhancer trap
Difference: enhancer is not transgenic but endogenous
Rescue experiments
A. Prokop
X-Gal4 ; UAS-lacZ
Experiments by group
Groups
larval genotype
staining
comments
BC
A-lacZ
2hr 37ºC, then
o.N. 26ºC
2 groups
DE
B-lacZ
2hr 37ºC
2 groups
FG
C-lacZ
2hr 26ºC
2 groups
HI
D-lacZ
2hr 37ºC, then
o.N. 26ºC
2 groups
KL
E-Gal4;UASlacZ
2hr 37ºC
2 groups, stock a bit weak
MN
F-Gal4;UAS-lacZ
2hr 37ºC
3 groups
OP
G-lacZ
2hr 37ºC, then
o.N. 26ºC
2 groups
A. Prokop
Performing enhancer trap screens
Experimental applications for enhancer-trap lines
perform
gain-of-function
experiments
and identify the gene
A. Prokop
use the line as
marker for
experiments
A. Prokop
Carry out loss-of-function studies
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