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

2. enhancer trap: expresses bGal in same pattern as target gene
P element constructs
enhancer trap: expresses bGal in same pattern as target gene
enhancer
target gene
lacZ
white

3. GAL4 enhancer trap: expresses Gal4p in same pattern as target gene
P element constructs
controlled misexpression: expresses target gene in Gal4-dependent manner
white
UAS
GAL4
white
GAL4 enhancer trap: expresses Gal4p in same pattern as target gene

4. insulators: block enhancers and position effects on expression
P element constructs
insulators: block enhancers and position effects on expression
white
yellow

5. Mapped P element Insertion Lines
(Bloomington Stock Center, as of 11/12/02)
P{PZ} enhancer trap
523
P{lacW} enhancer trap
1176
P{Gal4} Gal4 expression
141
P{EP} UAS-controlled expression
263
P{SUPor-P} insulator
2076
P{GT1} gene trap
511
4690

6. Transposition of P Elements
Sb D2-3 +
P transposase
(chromosome 3)
dominant
marker
w P{w+}
P element on
X chromosome
w P{w+}
Sb D2-3 + ; Y w
screen for
red-eyed sons w + ; Y
P{w+}
new insertion on autosome

7. P elements rarely insert into coding sequences
Spradling et al. (1995)

8. screen for loss of w+, indicating excision
Excision of P Elements
dominant
marker
P transposase
(chromosome 3)
P element on
chromosome 3
Sb D2-3
w ; P{w+} +
Sb D2-3
P{w+} w Y ; w w + ; Y
P{w+}**
screen for loss of w+, indicating excision

9. ...ATGCCAAACATGATGAAATAACATAAGGTGGTCCCGTCG...
...TACGGTTTGTACTACTTTATTGTATTCCACCAGGGCAGC...
31-bp P inverted repeat
8-bp target site
white
P transposase
P transposase
...ATGCCAAACATGATGAAATAACATA
...TACGGTTT
17-nt 3’ overhang

10. (double-strand break)
non-homologous
end-joining
homologous
recombination
different products, depending on: template for repair
extent of repair
gap widening before repair

11. 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

12. deletion of flanking DNA
C. “Imprecise excision”
exonuclease
repair
deletion of flanking DNA

13. find complementary mRNA
RNA Interference
dsRNA
Dicer endonuclease
21-23 bp (or nt) siRNA
find complementary mRNA
(RISC complex)
destroy mRNA

14. 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.

15. 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

16. 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)

17. 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

18. FLP Recombinase Catalyzes Exchange Between Target Sequences (FRTs)
crossover

19. Intrachromosomal Recombination Between Tandem FRTs
Results in Excision from the Chromosome
FRT
FRT
extrachromosomal circle
with 1 FRT
chromosome with 1 FRT

20. 5' ATTACCCTGTTATCCCTAAATT 3' 3' TAATGGGACAATAGGGATTTAA 5'
I-SceI makes a double-strand break at an 18-bp
target sequence
5' ATTACCCTGTTATCCCTAAATT 3'
3' TAATGGGACAATAGGGATTTAA 5'
5' ATTACCCTGTTAT CCCTAAATT 3'
3' TAATGGGAC AATAGGGATTTAA 5'
I-SceI

21. donor construct (integrated P element)
FRT
I-SceI site
FRT
donor construct (integrated P element)
FLP recombinase
extrachromosomal circular donor

22. I-SceI endonuclease
DSB * *
integration
tandem duplication

23. w+ *
FLP recombinase
I-SceI endonuclease * * w+
integration

24. * * * Tandem Duplications can be Reduced to Single Copy I-CreI site w+
I-CreI endonuclease * w+
DSB *
Repair of a DSB between direct repeats

25. 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