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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