#  Using TRiP lines 

 



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### TRiP constructs are injected into the following stocks

- older stock collections
    - *y sc v; attP2* (genotype: *y\[1\] sc\[1\] v\[1\]; P{y\[+t7.7\]=CaryP}attP2*)
    - *y sc v; attP40* (genotype: *y\[1\] sc\[1\] v\[1\]; P{y\[+t7.7\]=CaryP}attP40*)
- newer stock collections
    - *y v; attP2* (genotype: *y\[1\] v\[1\]; P{y\[+t7.7\]=CaryP}attP2*)
    - *y v; attP40* (genotype: *y\[1\] v\[1\]; P{y\[+t7.7\]=CaryP}attP40*)

These stocks can be used as controls, but other control lines are also available [here](/fly/trip-rnai-control-fly-stocks).

### TRiP stock phenotype

- wildtype cuticle color, *yellow+ (y+)* indicates that the attP site is present
- wildtype eye color, *vermillion+ (v+)* indicates that the hairpin is present
- loss of scutellar bristles*, scute- (sc-)* indicates the presence of the *y sc v* X chromosome
    - note that the TRiP no longer injects into *sc* mutant embryos
    - thus newer TRiP stocks lack the *sc* bristle phenotype

The genotype of each TRiP stock can be found in the downloadable excel sheet on the [stocks page](/fly/trip-rnai-fly-stocks).

Please note that TRiP stocks do not contain a mini-white marker. For the TRiP stocks that have recessive sc on the X chromosome, the allele of sc used is not very penetrant if grown at 18C, but is fully penetrant at 25C.

### Lethality

- 5-10% of the TRiP stocks are homozygous lethal
- lethal stocks are maintained over balancers
- we attribute lethality to naturally occurring second site lethals on the 2nd or 3rd chromosomes
- non-lethal but unhealthy stocks are also maintained over balancers to protect the integrity of the stock
- if the balancer is present in all flies, then 50% of the progeny will not carry the hairpin
- second site lethal can be recombined away from the attP insertion site if necessary
- when examining embryos, larvae or pupae, switch balancer to one containing marker present at the relevant stage of development

### Expressing TRiP lines

- any GAL4 line can be used to activate transcription of a TRiP hairpin under UAS control
- without a GAL4 driver present you should not get expression of the hairpin and thus you should not see any knock down phenotype
- if phenotype observed in an otherwise wildtype fly, then the transgene is somewhat "leaky" (this is rare)
- GAL4 drivers commonly used by the TRiP are listed in the [TRiP Toolbox](/fly/trip-toolbox) and can be obtained from the [BDSC](http://flystocks.bio.indiana.edu/Browse/TRiPtb.htm)
- visit the [RNAi Stock Validation & Phenotypes (RSVP)](/fly/rsvp) page for data on TRiP stock quality (knockdown ability, phenotypes, etc.)

###  Enhancing knockdown with UAS-dicer2

- drivers containing UAS-dicer2 are included in the [TRiP Toolbox](/fly/trip-toolbox)
- for TRiP stocks constructed in the VALIUM1 vector, maximum knockdown will only be attained if UAS-dicer2 is carried in the GAL4 driver
- TRiP stocks constructed in the VALIUM10, VALIUM20, VALIUM21 or VALIUM22 vectors do not require inclusion of UAS-dicer2 with the Gal4.
- to determine which VALIUM vector your TRiP stock uses please refer to the list of current TRiP stocks at the [TRiP RNAi fly stocks page](/fly/trip-rnai-fly-stocks)
- For expression of TRiP lines during oogenesis, see [GAL4-UAS in the germline](/fly/gal4-uas-germline)

###  Off target effects (OTE)

- short hairpin microRNAs in VALIUM20, VALIUM21 and VALIUM22 are not expected to have any OTE
- for long hairpins in VALIUM1 and VALIUM10 you can predict the off-targets by following the protocol below
    - Input your gene name into the [Updated Targets of RNAi Reagents (UP-TORR)](/fly/up-torr) search tool
    - Under “Off-target search options” select “Check off-target genes for matches greater than or equal to” and select the number of base pairs
    - Click submit, find the long hairpin line in the list of reagents, and click on the number in the OTE column.

### Feedback

- if a TRiP stock does not produce an expected phenotype please email the TRiP and/or report your result on the [RNAi Stock Validation Project (RSVP) feedback](/fly/rsvp-addfeedback) page and let us know
- ineffective stocks 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