#  GAL4-UAS in the germline 

 



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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](http://flystocks.bio.indiana.edu/Reports/31777.html)

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](http://www.ncbi.nlm.nih.gov/pubmed/17215303)

Stacy Mazzalupo and Lynn Cooley. 2006. "Illuminating the role of caspases during Drosophila oogenesis." Cell Death Differ 13, 1950-1959. PubMed ID: [16528381](http://www.ncbi.nlm.nih.gov/pubmed/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](http://www.ncbi.nlm.nih.gov/pubmed/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](http://www.ncbi.nlm.nih.gov/pubmed/10628987)

Pernille Rorth. 1998. "Gal4 in the Drosophila female germline." Mechanisms of development 78, 113-118. PubMed ID: [9858703](http://www.ncbi.nlm.nih.gov/pubmed/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](http://www.ncbi.nlm.nih.gov/pubmed/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](http://www.ncbi.nlm.nih.gov/pubmed/9118811)