Marek Mlodzik

LOCAL MUTAGENESIS BY IMPRECISE P-ELEMENT EXCISION

local mutagenesis; P-element excision;
This method is based on the fact that in most cases P-elements excise imprecisely when transposing via the P transposase. Most of these imprecise excision events leave sequences from the P elements behind. However, in a reasonable proportion of excisions (~10%) flanking genomic DNA is removed 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, e.g. in lines of the P[lacZ] insertions that do not have a phenotype by itself.
The following crossing schemes are using the «2-3 element at 99B as a source of the transposase. The given schemes are designed for P-elements with rosy ;as selectable marker. Nevertheless, very similar schemes can be used for other common markers such as the white; gene.

II. Chromosome P Insertions
1st generation:

MM mm
P[ry+] / P[ry+] ; ry / ry x CyO / Sp ; Sb, «2-3 / TM6, Ubx



2nd generation (here occurs the excision):

M (single males) mm
P[ry+] / CyO ; Sb, «2-3 / ry x CyO / Sco ; ry / ry



3rd generation:

M (single males, ry eye color) mm
E{P} / CyO ; ry / ry x CyO / Sco ; ry /ry



4th generation (brother-sister cross):

MM mm
E{P} / CyO ; ry / ry x E{P} / CyO ; ry / ry

established stock, if excision is lethal; check homozygous flies for visible phenotypes.

III. Chromosome P Insertions

1st generation:

MM mm
P[ry+] , ry / P[ry+] , ry x Ki, pp, «2-3 / Ki, pp, «2-3



2nd generation (here occurs the excision):

M (single males) mm
P[ry+] , ry / Ki, pp, «2-3 x TM3, ry / rf10 (ftz, e, ry)



3rd generation:

M (single males, ry eye color) mm
E{P}, ry / TM3, ry x TM3, ry / rf10



4th generation (brother-sister cross):

MM mm
E{P}, ry / TM3, ry x E{P}, ry / TM3, ry


established stock, if lethal; check for visible mutations in homozygous flies


X Chromosome P Insertions:

1st generation:

mm MM
P[ry+] / P[ry+] ; ; ry / ry x + / -- ; ; Ki, pp, «2-3/ Ki, pp, «2-3


2nd generation (here occurs the excision):

M (single males) mm
P[ry+] / -- ; ; Ki, pp, «2-3 / ry x FM6 / dnd ; ; ry / ry



3rd generation:

m (single virgins, ry eye color) MM
E{P} / FM6 ; ; ry / ry x FM6 / -- ; ; ry / ry


established balanced stock, if excision is lethal; check for visible phenotypes in homozygous flies.