Transcript Slide 1

Reverse Genetics in Drosophila
I. P elements in reverse genetics
A. P element insertional mutagenesis projects
B. Using P elements to make mutations
II. RNA interference
A. Basics of RNAi
B. RNAi methods in flies
III. Targeted gene replacement
P element constructs
enhancer trap: expresses bGal in same pattern as target gene
enhancer
lacZ
target gene
white
P element constructs
controlled misexpression: expresses target gene in Gal4dependent manner
white
GAL4
UAS
white
GAL4 enhancer trap: expresses Gal4p in same pattern as target gene
P element constructs
insulators: block enhancers and position effects on expression
white
yellow
Mapped P element Insertion Lines
(Bloomington Stock Center, as of 11/12/02)
P{PZ} enhancer trap
P{lacW} enhancer trap
523
1176
P{Gal4} Gal4 expression
141
P{EP} UAS-controlled expression
263
P{SUPor-P} insulator
P{GT1} gene trap
2076
511
4690
Transposition of P Elements
dominant P transposase
marker (chromosome 3)
w P{w+}
P element on
X chromosome
Sb D2-3
+
w P{w+} ; Sb D2-3
Y
+
w
screen for
red-eyed sons
w
Y
P{w+}
;
+
new insertion on autosome
P elements rarely insert into coding sequences
Spradling et al. (1995)
Excision of P Elements
dominant P transposase
marker (chromosome 3)
P element on
chromosome 3
w ; P{w+}
w
Y
;
Sb D2-3
+
P{w+}
w
Sb D2-3
w
Y
P{w+}**
;
+
screen for loss of w+, indicating excision
white
P transposase
8-bp target site
31-bp P inverted repeat
...ATGCCAAACATGATGAAATAACATAAGGTGGTCCCGTCG...
...TACGGTTTGTACTACTTTATTGTATTCCACCAGGGCAGC...
P transposase
...ATGCCAAACATGATGAAATAACATA
...TACGGTTT
17-nt 3’ overhang
(double-strand break)
non-homologous
end-joining
homologous
recombination
different products, depending on:
template for repair
extent of repair
gap widening before repair
A. Repair using sister chromatid as a template
white
restoration of P element (w+)
whi
internal deletion of P element (w-)
sometimes alters expression of target gene
B. Repair using homologous chromosome as a template
precise excision
useful for proving that phenotypes are due to P element insertion
C. “Imprecise excision”
exonuclease
repair
deletion of flanking DNA
RNA Interference
dsRNA
Dicer endonuclease
21-23 bp (or nt) siRNA
find complementary mRNA
(RISC complex)
destroy mRNA
Functions for RNA Interference
Repression of repeated genes (e.g., transposable elements)
Defense against viruses (plants)
Developmental control of gene expression (small temporal RNAs)
X chromosome inactivation (mammals)
Silencing of mating type loci and centromeric regions (S. pombe)
DNA elimination in macronuclei (Tetrahymena)
Experimental manipulation of gene function.
RNAi Methods in Drosophila
1. Addition of dsRNA to cell culture
2. Injection of dsRNA into embryos
3. Expression of hairpin RNA in vivo.
UAS
Gal4
RNA
dsRNA
Gene Targeting Technologies
S. cerevisiae
Generate linear targeting DNA by PCR
Transform suitable strain
Plate on medium for positive selection (10-8?)
M. musculus
Generate targeting DNA by cloning, cutting
Transform ES cells
Conduct positive and negative selections (typical = 10-7)
Gene Targeting in Drosophila
Problems
No culture system for germline stem cells
DNA introduced by injection in single embryos
Existence of DNA repair in early development questionable
Solution (Rong and Golic)
Generate linear DNA in vivo:
Obtain stable transformants of donor construct
Use FLP – FRT system to excise donor DNA from chromosome
Use I-SceI to linearize donor DNA
Use visible marker gene to screen for potential homologous
gene replacements
FLP Recombinase Catalyzes Exchange Between
Target Sequences (FRTs)
FRT
FLP recombinase
crossover
Intrachromosomal Recombination Between Tandem FRTs
Results in Excision from the Chromosome
FRT
FRT
extrachromosomal circle
with 1 FRT
chromosome with 1 FRT
I-SceI makes a double-strand break at an 18-bp
target sequence
5' ATTACCCTGTTATCCCTAAATT 3'
3' TAATGGGACAATAGGGATTTAA 5'
I-SceI
5' ATTACCCTGTTAT
3' TAATGGGAC
CCCTAAATT 3'
AATAGGGATTTAA 5'
FRT
FRT
I-SceI site
donor construct
(integrated P element)
FLP recombinase
extrachromosomal
circular donor
I-SceI endonuclease
DSB
*
integration
*
tandem duplication
w+
*
FLP recombinase
I-SceI endonuclease
*
integration
w+
*
Tandem Duplications can be Reduced to Single Copy
I-CreI site
w+
*
I-CreI endonuclease
w+
*
DSB
Repair of a DSB between direct repeats
*
Reverse Genetics in Drosophila
I. P elements in reverse genetics
A. P element insertional mutagenesis projects
B. Using P elements to make mutations
II. RNA interference
A. Basics of RNAi
B. RNAi methods in flies
III. Targeted gene replacement