Genetic Transformation of Drosophila with Transposable Element Vectors SCIENCE, VOL. 218, 22 OCTOBER 1982
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1 Genetic Transformation of Drosophila with Transposable Element Vectors SCIENCE, VOL. 218, 22 OCTOBER 1982 Transposition of Cloned P Elements into Drosophila Germ Line Chromosomes SCIENCE, VOL. 218, 22 OCTOBER 1982 Gerald M. Rubin and Allan C. Spradling
2 Outline Introduction General Information Experiments Results Take-home message Questions
3 Drosophila melanogaster
4 Drosophila melanogaster completly sequenced in the year 2000 Genome size: about million bp, genes Good model organism for genetics (small, short generation time, easy to obtain) 75% of the genes are homologous to human disease genes
5 DNA Transposons DNA segments which are capable of changing their position within the genome of a cell In D. melanogaster 15-22% of the genom are transposable elements (in human genom: about 45%) Limited by small inverted-repeat sequences which are essential for transposase activity
6 Transposase
7 Transposase
8 P-element DNA-Transposon in Drosophila melanogaster Length: 2907bp, contains on both termini 2 inverted repeats (31 bp) 4 exons which code for the Transposase
9 P-element
10 Hybrid dysgenesis
11 Hybrid dysgenesis Phenotype occurs when P-type males are mated with M-type females Egg cells of the females have no repressor for transposase Transposase causes high mutational rate Progeny is sterile
12 Experiment-1 Mimicking the H.D. Assumptions: P-Factors enter the cell with the sperm micro-injection of pπ25.1,a plasmid containing the 3kb P-element All progeny has to be tested for the injected DNA mutation with distinguishable phenotype
13 Allele of choice: singed(sn) Controls morphology of the bristles sn w -singed weak (result of hybride dysgenesis, located on the X-chromosome) sn w wildtype sn +
14 Experimental design Conclusion: sn w is not able to catalyze transposition itself
15 Digestion with SalI/ HindIII Hybridization with appropriate 1.5/8.4 kb fragments of pπ25.1 unstable lines contain at least one complete P factor
16 Experiment-2: Localization of P-element integration sites Isolation of polytene chromosomes of line 301 Polytene chromosomes Giant chromosomes Result of replication without following cell division Sister chromatids synapsed together can be seen with a microscope
17 In situ hybridization with 3 H-labeled RNA complementary to pπ25.1 sn w M strain sn w locus Flanking sequences of the P-element in pπ25.1 Additional site in
18 Experiment-3: P-element based vectors used for genetic transformation 2 possibilities: Using a 3 kb P Element as vector by integrating gen of interest Problem: integration sites must be known, not to destroy important sequences Inserting the gen of interest into a 1,2 kb P- element, not able of transposition
19 rosy gene (ry + ) encodes for Xanthine Dehydrogenase Reason for this choice: A cloned DNA segment containing ry + was available Causes visible and selectable phenotypes Normal eye colour is restored by 5% XDH-activity XDH has not to be present in eye cells
20 8.1 kb Sal I DNA fragment Xho I restriction site rosy ry + gen flanked by 1,2 kb P- element lacking the ability to transpose pry1/pry3 (difference: orientation of the 8.1 kb Sal I sequence)
21
22 Verification Experiments Hybridization with pdm2844s8.5 additional ry + genes Hybridization with pm12.8 no additional sequences existing
23 Summary P-elements are the transposable elements in Drosophila melanogaster, they code for the Transposase defective progeny occurs only when P-males are mated with M-females (Hybrid dysgenesis) Polytene-chromosomes can be used to map the P-element insertion sites Transgene strains of D. melanogaster can be generated by using two plasmids, one containing the P-element
24 Take Home Message Transposable Element (P-element) vectors can be used to modify the genome of Drosophila melanogaster and so it could be used to modify other animals or plants as well.
25 Questions 1. Wie werden transgene Drosophila-Stämme mit Hilfe von P- Elementen erzeugt? Beschreiben Sie die Methode. 2. Was sind Polytänchromosomen und wie können sie beim Kartieren von Transgen-Insertionen benützt werden?
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