GAL4-UAS in the germline

Second generation VALIUM22 and VALIUM21, and to a lesser extent VALIUM20, shRNA vectors allow effective knockdown during oogenesis, providing an alternative to producing germline clones.

The TRiP recommends the MTD-GAL4 driver for maternal and germline expression of shRNAs

The Maternal Triple Driver (MTD)-GAL4

  • homozygous insertions of three GAL4 constructs
    • P{COG-GAL4:VP16}
      • made by Pernille Rorth (Rorth, 1998)
      • pCOG vector (Robinson and Cooley, 1997) contains a promoter from the otu gene and the 3’ UTR from the K10 gene
      • GAL4:VP16 expression from this transgene is weak or absent in the germarium, and robust beginning in stage 1 egg chambers (Rorth, 1998)
    • P{nos-GAL4-VP16}
      • contains both the promoter and 3’ UTR from the nanos gene (Van Doren et al., 1998)
      • made to achieve maternal loading of GAL4 in order to analyze translation control in pole cells
      • Expressed in ovaries throughout the germarium and in all stages of egg chambers
      • somewhat lower expression in very young egg chambers (~stages 2-6) (Rorth, 1998)
    • P{GAL4-nos.NGT}40
      • contains the nanos promoter and aTub84E 3’ UTR (Tracey et al., 2000)
      • made for maternal loading of Gal4 to drive expression during embryogenesis.
  • together provide robust germline and maternal GAL4 expression
  • genotype is P{COG-GAL4:VP16}; P{Gal4-nos.NGT}40; P{nos-Gal4-VP16}
  • Bloomington stock #31777 

Combinations of these transgenes were first reported by Grieder et al. (Grieder et al., 2000) in their effort to produce uniform expression of Tubulin:GFP in the germarium and in egg chambers. Andrew Hudson in the Cooley lab established a stable homozygous stock dubbed MTD-Gal4, which was published in papers examining germline caspase activity (Mazzalupo and Cooley, 2006) and the function of the ring canal protein made by the hts gene (Petrella et al., 2007).

Relevant publications

Lisa Petrella, Tracy Smith-Leiker, and Lynn Cooley. 2007. "The Ovhts polyprotein is cleaved to produce fusome and ring canal proteins required for Drosophila oogenesis." Development 134, 703-712. PubMed ID: 17215303

Stacy Mazzalupo and Lynn Cooley. 2006. "Illuminating the role of caspases during Drosophila oogenesis." Cell Death Differ 13, 1950-1959. PubMed ID: 16528381 

Nicole Grieder, Margaret de Cuevas, and Allan Spradling. 2000. "The fusome organizes the microtubule network during oocyte differentiation in Drosophila." Development 127, 4253-4264. PubMed ID: 10976056

William Tracey, Jr., Xiangqun Ning, Martin Klingler, Sunita Kramer, and Peter Gergen. 2000. "Quantitative analysis of gene function in the Drosophila embryo." Genetics 154, 273-284. PubMed ID: 10628987 

Pernille Rorth. 1998. "Gal4 in the Drosophila female germline." Mechanisms of development 78, 113-118. PubMed ID: 9858703

Mark Van Doren, Anne Williamson, and Ruth Lehmann. 1998. "Regulation of zygotic gene expression in Drosophila primordial germ cells." Curr Biol 8, 243-246. PubMed ID: 9501989

Douglas Robinson and Lynn Cooley. 1997. "Examination of the function of two kelch proteins generated by stop codon suppression." Development 124, 1405-1417. PubMed ID: 9118811