Transcript P-elements and Enhancer Trapping Naturally occurring P

Expt. 10-1: P-elements and
Enhancer Trapping
Expt. 10: P-elements and
Enhancer Trapping
-Naturally occurring P-elements:
Transposase gene between two inverted repeats
Transposase
Organization of the P Element
Transposase ORF
2.9 kb P Element
31 bp Inverted
Repeat
Tnp Binding
Site
9 or 21 bp Spacer
Transposase catalyzes a 17bp staggered cleavage event
5’ Cleavage Site
NNNNNNNNNCATGATGAAATAACATAAGGTGG
NNNNNNNNNGTACTACTTTATTGTATTCCACC
3’ Cleavage Site
NNNNNNNNNCATGATGAAATAACATA
NNNNNNNNN 5’-P
3’-OH
AGGTGG
HO-3’ GTACTACTTTATTGTATTCCACC
P -5’
P Element
+
P element integration generates an 8 bp target site duplication
8 bp target site
duplication
P Element
Taking Advantage of P-elements
-Mutagenesis
Taking Advantage of P-elements
-Mutagenesis
-Insertional mutations easy
to clone.
Taking Advantage of P-elements
-Mutagenesis
Insertional mutations (easy to clone).
-Imprecise excisions lead to frameshifts
-and deletions.
Taking Advantage of P-elements
-Germline transformation
Taking Advantage of P-elements
-Mutagenesis
-Germline transformation
1.Design transgene with
inverted repeats.
Taking Advantage of P-elements
-Mutagenesis
-Germline transformation
1.Design transgene with
inverted repeats.
2.Mix with transposase gene
Taking Advantage of P-elements
-Mutagenesis
-Germline transformation
1.Design transgene with
inverted repeats.
2.Mix with transposase gene
3.Inject into germline of embryo
Taking Advantage of P-elements
Germline transformation
1.Design transgene with
inverted repeats.
YFG
2.Mix with source of transposase
3.Inject into germline of embryo
4.Look for transformants in F1.
Taking Advantage of P-elements
-Mutagenesis
-Germline transformation
-Enhancer trapping
Use regulatory information
from nearby genes to drive
expression of a transgene
Mechanics of Enhancer Trapping:
-Modified P-element contains:
-Inverted repeats
-Basic promoter sequences
-Molecular marker gene (e.g. b-galactosidase)
-Phenotypic marker (e.g. w+, ry+)
-Mobilize using transposase
-Confirm hopping in F1 (phenotype)
-Look for interesting/desired expression pattern in F2 with lacZ staining
P
lacZ
w+
Mechanics of Enhancer Trapping:
-Modified P-element contains:
-Inverted repeats
-Basic promoter sequences
-Molecular marker gene (e.g. b-galactosidase)
-Phenotypic marker (e.g. w+, ry+)
-Mobilize using transposase
-Confirm hopping in F1 (phenotype)
-Look for interesting/desired expression pattern in F2 with lacZ staining
ey enhancer
P
UAS
lacZ
GAL4
w+
UAS
InR(DN)
Attached X females
XY
X
^
XXY
Attached X females
XY
^
XXX
^
XXY
XY
YY
X
^
XXY
Sterile
Female
Male
Dead
+ P[lacZ, ry+], Cy ry
+
Sco
ry
X
+ + Ki D2-3, ry+
Y +
+
+ P[lacZ, ry+], Cy ry
+
Sco
ry
X
+ + Ki D2-3, ry+
Y +
+
+ P[lacZ, ry+], Cy Ki D2-3, ry+
Y
+
ry
(phenotype=Cy, Ki, ry+)
XX + ry
Y + ry
+ P[lacZ, ry+], Cy ry
+
Sco
ry
X
+ P[lacZ,
Cy Ki D2-3,
Y
+
ry
(phenotype Cy, Ki, ry+)
ry+],
+ + Ki D2-3, ry+
Y +
+
ry+
X
^
^
XX + ry
Y + ry
+ P[lacZ, ry+], Cy ry
+
Sco
ry
X
+ P[lacZ,
Cy Ki D2-3,
Y
+
ry
(Cy, Ki, ry+)
ry+],
+P?
+P?
+ + Ki D2-3, ry+
Y +
+
ry+
ryP?
Y
+
ry
(not Cy, not Ki. If carrying
a jumped P, will be ry+)
X
^
XX + ry
Y + ry
^
XX +
Y +
ry
ry
+ P[lacZ, ry+], Cy ry
+
Sco
ry
+ + Ki D2-3, ry+
Y +
+
X
+ P[lacZ,
Cy Ki D2-3,
Y
+
ry
(Cy, Ki, ry+)
ry+],
+P?
+P?
ryP?
Y
+
ry
ry+
X
X
^
XX + ry
Y + ry
^
XX +
Y +
(not Cy, not Ki. If carrying
a jumped P, will be ry+)
Stain F2 for lacZ
Look for desired
expression pattern
ry
ry
We want to look at
enhancer traps that
express in the ovaries.
The Ovariole
Germarium
Germarium
The Ovariole
The Egg Chamber
Nurse Cells
Oocyte,
follicle cells
Staining Ovaries
Schematic Summary
-Dissect ovaries out of abdomen in NaPO4 buffer (movie!).
-Devitallinize with heptane
-Fix ovaries and wash
-Add X-GAL, the substrate for b-galactosidase.
-Wash when dark enough, and observe.
-We are using enhancer traps that express in
-Follicle cells
-Nurse cells and oocyte