Transgenes


site-specific insertion using the attB/phi-C31 system

Transgenes are inserted at a selected site of the genome by the integrase phi-C31 using attB sites. The system offers high integration rates (upto 1 in two lines), size-independent integration rate, no need to map the insertion site and comparable expression levels of different transgenes. A describtion of the system is available at http://www.frontiers-in-genetics.org/flyc31/ The transgenic contruct is injected into embryos with the target site and phi-C31 transgene. Injection should be at 0.1 ug/ul, higher concentrations may result in unspecific insertions. It is highly recomended to cross out the phi-C31 integrase after selecting the transgene.

Procedure:


  1. preparation of a plasmid with 5'P and 3'P,  e. g. Casper
  2. precipitate about 3 µg of plasmid with 1µg of Delta2-3turbo (transposase) dissolve DNA in water to 0,2 to 0,6 µg/µl.
  3. inject DNA into w embryos prior to pole cell formation, deposit DNA in the pole plasma at the posterior tip of the embryo
  4. cultivate the injected embryos  (G0 generation), transfer hatched larvae from the oil to a yeasted apple juice plate. When pupation starts transfer the 3rd instar larvae and pupae to a small food vial with a cardboard in it. Keep the vial in humid conditions.
  5. cross G0 adults with yw  virgins or males.  use 2 G0 males x 3 yw virgins  and 3 G0 females with 3 to 5 yw males, use yeasted food vials, flip over  to new yeasted vials after 3 to 4 days. Add filter, if the food gets to fluid. If you have a sufficient number of flies you may already use the double balancer stock at this stage to shorten the scheme by one generation.
  6. screen the F1 flies for  yellow/orange/red eyes every day. w+ flies from the same vial may originate from the same insertion event. pick one male (or female) per vial, keep other w+ flies in reserve
  7. cross the F1 w+ male (or female) with 3 females (males) of w; Sp / CyO;  Dr / TM3, Sb, flip the cross into a yeast food vial after 3 to 4 days, add appropriate amounts of yeast to the vial
  8. cross one w+ F2 male, w; +/CyO;  +/TM3 with w; TM3/TM6B to test the segration with TM3, cross similar w+ F2 male with w; Tft / CyO to test the segrgation with CyO
  9. decide on which chromosome the w+ transgene has integrated,
    • on X:  the Y chromosome and the w+ segregate:   all females are w+
    • on III:   the w+ and the TM segregate:  all TM3/TM6 flies are w
    • on II:  if conditions 1 and 2 are not true.
    • on IV:  hardly ever the case
  10. balancing of the insertion:
    • III.  cross a male w+ /TM3 with w; TM3/TM6B virgins
    • II:  cross a male w+ with w; Tft/CyO virgins, collect w+/CyO males and females in the next generation
    • X:  cross w+ virgins with FM7 males, collect w+/FM7 and w+ males in the next generation


Stocks:   
y w
w; Sp / CyO;  Dr / TM3, Sb
w;  TM3, Sb / TM6B, Hu Tb
w;  Tft / CyO