The TRiP stocks are in a y sc v; attP2 (genotype: y[1] sc[1] v[1]; P{y[+t7.7]=CaryP}attP2) or y sc v; attP40 genetic background (genotype: y[1] sc[1] v[1]; P{y[+t7.7]=CaryP}attP40).

These stocks can be used as controls, but other control lines are also available here.

A. The control lines that people often use are simply TRiP lines which are known NOT to function in the process or pathway being studied. However, you can also use VALIUM10-GFP or VALIUM10-luciferase for TRiP lines in VALIUM10 and VALIUM20-GFP and VALIUM20-luciferase for TRiP lines in VALIUM20.

See our list of suggested control lines here.

The attP docking site of the TRiP stocks is in either attP2 located on the third chromosome at 68A4 or attP40 located on the second chromosome at 25C6.

In the final homozygous TRiP lines y+ indicates that the attP site is present, v+ indicates that the hairpin is present, and sc- indicates the presence of the y sc v X chromosome. All of which confirm the correct genotype.

All TRiP stocks have a wildtype appearance as the wildtype vermillion gene is used as the selection marker for the hairpin transformants and the attP insertions carry the wildtype yellow gene.

No TRiP stocks contain "mini-white".

About 5-10% of the TRiP lines are homozygous lethal. We attribute this to second site lethals on the 2nd or 3rd chromosomes which occur naturally in lab stocks over time. In some cases this second mutation may not be entirely lethal, and homozygotes can be obtained for your experiments, but the balancer (TM3 or CyO) is maintained and protects the integrity of the stock. If, however, the balancer is present in all flies, then you will simply have to accept that 50% of the progeny will not carry the hairpin. Alternatively you could try to recombine away the second site lethal from the attP insertion site. If you are examining embryos, larvae or pupae you might want to change the balancer to one that can be followed with lacZ or another marker present at the relevant stage of development.

Many of the TRiP lines have been tested for their "knock-down" abilities. Since this initial testing, however, the TRiP has entered the "production phase" where not all lines are being tested.

The GAL4 drivers commonly used by the TRiP are listed in the TRiP Toolbox. Some of these drivers also contain UAS-dicer2, which enhances the knock-down. These lines can be obtained from the BDSC.

The Maternal Triple Driver (MTD)-Gal4 stock contains homozygous insertions of three Gal4 constructs, which together provide robust germline and maternal Gal4 expression. The genotype is P{COG-GAL4:VP16}; P{Gal4-nos.NGT}40; P{nos-Gal4-VP16} (Bloomington stock #31777).

• P{COG-GAL4:VP16} was made by Pernille Rørth as part of her effort to establish the Gal4/UAS system in the germline (Rorth, 1998). It was made in the pCOG vector (Robinson and Cooley, 1997) that 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). It was made to achieve maternal loading of Gal4 in order to analyze translation control in pole cells. In ovaries, it is expressed throughout the germarium and in all stages of egg chambers, with 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), and was made for maternal loading of Gal4 to drive expression during embryogenesis.

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).

References:

Grieder, N.C., de Cuevas, M., and Spradling, A.C. (2000). The fusome organizes the microtubule network during oocyte differentiation in Drosophila. Development (Cambridge, England) 127, 4253-4264.

Mazzalupo, S., and Cooley, L. (2006). Illuminating the role of caspases during Drosophila oogenesis. Cell Death Differ 13, 1950-1959.

Petrella, L.N., Smith-Leiker, T., and Cooley, L. (2007). The Ovhts polyprotein is cleaved to produce fusome and ring canal proteins required for Drosophila oogenesis. Development (Cambridge, England) 134, 703-712.

Robinson, D.N., and Cooley, L. (1997). Examination of the function of two kelch proteins generated by stop codon suppression. Development (Cambridge, England) 124, 1405-1417.

Rorth, P. (1998). Gal4 in the Drosophila female germline. Mechanisms of development 78, 113-118.

Tracey, W.D., Jr., Ning, X., Klingler, M., Kramer, S.G., and Gergen, J.P. (2000). Quantitative analysis of gene function in the Drosophila embryo. Genetics 154, 273-284.

Van Doren, M., Williamson, A.L., and Lehmann, R. (1998). Regulation of zygotic gene expression in Drosophila primordial germ cells. Curr Biol 8, 243-246.

If the TRiP stock you are using was constructed in the VALIUM10, VALIUM20, VALIUM21 or VALIUM22 vector, it is likely that UAS-dicer2 will not be necessary to achieve maximum knock-down of your target gene.

However, if the TRiP stock you are using was constructed in the VALIUM1 vector, it is essential that UAS-dicer2 be carried in the GAL4 driver you are using.

To determine which VALIUM vector your TRiP stock utilized please refer to the list of available TRiP stocks.

Without a GAL4 driver present you should not get expression of the hairpin and thus you should not see any knock down phenotype. If you are seeing the phenotype in an otherwise wildtype fly, then the transgene is somewhat "leaky", a situation that we have seen only one or two times before.

We welcome feedback!

If a TRiP stock does not produce an expected phenotype please email the TRiP and let us know. The construct will be removed from the collection and tested by the TRiP team. If sufficient knockdown is not observed, a new set of oligos will be designed and a new construct generated to replace the TRiP stock.

Our short hairpin microRNAs in VALIUM20, VALIUM21 and VALIUM22 are not expected to have any off-target effects. For long hairpins in VALIUM1 and VALIUM10 you can predict the off-targets by following the protocol below.

To find OTE information for 1st generation (long hairpin) TRiP stocks:

1. Retrieve the target sequence of TRiP stock of interest from the FlyBase website (http://flybase.org) by typing “dsRNA-stockid” in the quick search box. The target sequence can be found under the section titled "Sequence Data"
Example: For TRiP stock HM04026, enter “dsRNA-HM04026." (http://flybase.org/reports/FBsf0000151406.html ).

2. Copy the target sequence and run the “find OTEs” program (http://www.flyrnai.org/RNAi_find_frag_free.html) from the DRSC website. You can specify OTE size (16-50bp) and the default is 19bp. This program takes the input sequence in FASTA format, then identifies the number and the locations of OTEs, and finally locates the OTE-free regions within input sequence.

VALIUM20 and VALIUM22 have NheI and EcoRI restriction sites, and we use both sites to clone the 71bp shRNA into the vector.

We tested these mismatches and found they gave similar phenotypes as non-mismatches.

There's no need to purify these oligos. Even without purification you should get about 90% correct colonies after ligation and transformation.

To check the presence of shRNA in VALIUM20, the primers are:

F: 5’-ACCAGCAACCAAGTAAATCAAC-3’
R: 5’-TAATCGTGTGTGATGCCTACC-3’

The size of the PCR product is around 350bp.

To check the presence of shRNA in VALIUM22, the primers are:

F: 5’-ACCAGCAACCAAGTAAATCAAC-3’
R: 5’-GGTGATAGAGCCTGAACCAG-3’

The size of the PCR product is around 350bp.

To check the orientation you can use this primer from the ftz region of the WALIUM10 vector:
5’-TCTAGTTCTTTGCAATCTGTAAGCA-3’

Oligos used to design the RNAi hairpin are not appropriate for RT-PCR. For best results you should design one primer from the targeted cDNA and another primer from outside of the targeted region.

Injection of TRiP hairpin containing constructs is performed by Genetic Services, Inc. (GSI).

Genetic Services, Inc., One Kendall Square, Bldg. 300 Cambridge, MA 02139 E-mail: info@geneticservices.com

If use of any of the TRiP stocks or reagents results in data that is included in a manuscript for publication, the TRiP requests that a version of the following statement be included in the Acknowledgements section: "We thank the TRiP at Harvard Medical School (NIH/NIGMS R01-GM084947) for providing transgenic RNAi fly stocks and/or plasmid vectors used in this study."