Protocol - Local mutagenesis by imprecise P-element excision
It allows to create small deletions around the original P element insertion point.
Steps
| Description | Temperature | Time | Note |
|---|---|---|---|
|
Cross males P(ry+)/P(ry+); ry/ry with virgin females CyO/Sp; Sb, Delta2-3/TM6,Ubx
|
25°C
|
10 days
|
This crossing scheme is using the Delta 2-3 element at 99B as a source of the transposase and is designed for P-elements with rosy as selectable marker, inserted on II chromosome
|
|
Select F1 males: P(ry+)/CyO;Sb, Delta2-3/ry
|
RT
|
2 days
|
|
|
Collect virgin females CyO/Sco; ry/ry
|
RT
|
3 days
|
|
|
Cross single males P(ry+)/CyO;Sb, Delta2-3/ry with virgin females CyO/Sco; ry/ry
|
25°C
|
10 days
|
Consider that about 10% of excisions lead to remove flanking genomic DNA
|
|
Collect males with ry eye color: E(P)/CyO; ry/ry
|
RT
|
6-8 days
|
These males lost P element
|
|
Collect virgin females CyO/Sco; ry/ry
|
RT
|
3 days
|
|
|
Cross single males E(P)/CyO;ry/ry with virgin females CyO/Sco; ry/ry
|
25°C
|
10 days
|
|
|
Collect males and females E(P)/CyO;ry/ry from each lines
|
RT
|
6-8 days
|
|
|
Set up brother-sister cross.
|
Establish stock if excision is lethal; check homozygous flies for visible phenotypes
|
Other informations
This approach relies on the fact that in about 10% of excisions (~), P-elements excise imprecisely when transposing via the P transposase; these events remove flanking genomic DNA with the P elements, creating small deletions around the original P element insertion point.
The use of this approach allows to create local mutations in genes neighboring the original P insertion, as well as in lines of the P[lacZ] insertions that do not have a phenotype by itself.
The crossing schemes of the reported protocol are designed for P-elements with rosy as selectable marker, but similar schemes can be used for other traceable markers like the white gene. Moreover, the Delta2-3 element at 99B as a source of the transposase
In our work, we used the imprecise excision of the 9d1 PZ element at the 25E1–E3 polytene region to induce genomic deletions surrounding the insertion site, and to study the clot gene of D. melanogaster
Validation info
The protocol has been both published and used to obtain data in peer-reviewed journals.
Funded by: Grants from the Regione Campania (L.R. 31.12.1994 n 41) and the Consiglio Nazionale delle Ricerche (Cluster n 02 Legge 488/92) to M.FURIA
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Anesthetizing D. melanogaster.pdf
(application/pdf
25Kb)
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Collecting D. melanogaster virgins.pdf
(application/pdf
52Kb)
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Preparation of growth medium for D. melanogaster.pdf
(application/pdf
54Kb)
