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

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

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

insulators: block enhancers and position effects on expression 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 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

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

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

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

...ATGCCAAACATGATGAAATAACATAAGGTGGTCCCGTCG... ...TACGGTTTGTACTACTTTATTGTATTCCACCAGGGCAGC... 31-bp P inverted repeat 8-bp target site white P transposase 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

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

find complementary mRNA 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) crossover

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

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

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

I-SceI endonuclease DSB * * integration tandem duplication

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

* * * 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