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Q-SYSTEM

Repressible binary expression system for genetic labeling and manipulations.

Q-system

Available Q system reagents - Flies (including QF2) 

Available Q system reagents - DNA (including QF2) 

Figure 1 (click to enlarge)


Q-system

 

The “Q-system” consists of three components: the QF transcription factor, the QS suppressor, and a QUAS-effector (Figure 1). The QUAS element contains 5 QF binding sites, and allows for robust QF-dependent expression of the effector.

We recently modified the QF transcription factor so that it is no longer toxic when broadly expressed.  This new version is called QF2. Details can be found in Riabinina, 2015. 

 

Intersectional expression

 

Figure 2 (click to enlarge)

By using the Q-system with the GAL4 system, effector gene expression can be limited to subsets of tissues or expression patterns not previously possible. In this case, the interaction between the QF expression pattern and the GAL4 expression pattern can be thought of as logical operations (Figure 2). For example, limiting expression to where only QF AND GAL4 are expressing is an “AND” logic gate . Alternatively, one could use any of the thousands of GAL4 lines available to carve out expression of a QF line. This would be an example of QF NOT GAL4 where expression is limited to where QF is expressed, but NOT where GAL4 is expressed. Conversely, one could use a QF line to carve out expression of a GAL4 line (GAL4 NOT QF). The genetic strategies for such expression pattern manipulations are summarized in the Figure 2.

 

 

 

Mosaic Analysis

By using differential expression of the QS suppressor, the Q-system can also be used for Mosaic Analysis with a Repressible Cell Marker: Q-MARCM (Movie). This is analogous to the GAL4 based MARCM system (Lee and Luo, 1999), yet requires no GAL4 components. In this technique, loss of the QS suppressor is directed by a mitotic recombination event that segregates the QS suppressor into one progenitor cell but not the other (see Movie). Since the GAL4 and Q-systems work independently in vivo, the GAL4 system could be used to inhibit function in a population of cells, and the Q-system could be used to determine the effect of such GAL4 mediated perturbations on a single neuron or cell. For example. GAL4 could be used to drive UAS-RNAi library expression in a target tissue, and the Q-system could be used to visualize the targeting of a single neuron to the target tissue in the background of such RNAi expression. Therefore, the use of the GAL4 and Q-systems together make investigations into non-autonomous effects more feasible.

Figure 3 (click to enlarge)

Since the Q-system and GAL4 system function independently in vivo, Q-MARCM and GAL4 MARCM can be coupled to the same mitotic event. As such, an unlabeled progenitor cell would give rise by mitosis to one cell that is positively labeled by the Q system (as it lacks the QS repressor), and a sister cell that is positively labeled by the GAL4 system (as it lacks the GAL80 repressor) (Movie ). This is called “coupled MARCM” as the segregation of the QS and GAL80 suppressor are coupled to the same mitotic event (Figure 3). 

For any questions regarding the Q-system, contact Chris Potter cpotter@jhmi.edu

Splinkerette PCR

Figure 1 (click to enlarge)Figure 2 (click to enlarge)

FRT Map Positions are available here.
The splinkerette protocol is available here.


In Drosophila, the most commonly used methods for introducing a transgene into the genome is mediated by the P-element transposon (Rubin and Spradling, 1982; Spradling and Rubin, 1982) or the piggyBac transposon (Handler et al., 1999). In these approaches, the transgene to be integrated is flanked by P-element or piggyBac transposable elements ends, which can integrate the transgene into the germline in the presence of a transposase enzyme. The result is a transgene inserted into the genome flanked by transposable element ends.

It is often useful to determine the exact genomic insertion site for the transgene. An approach for mapping insertion sites is splinkerette PCR (spPCR) (Figure 1). This technique was originally developed to amplify the genomic DNA between a known restriction site and a target gene (Devon et al., 1995), and then adapted to map the insertion sites of viral integrating gene traps in the mouse genome (Horn et al., 2007). In this technique, genomic DNA is digested to yield overhanging sticky ends (Figure 1). The restriction enzyme is not required to cut within the transgene. Onto this sticky end is ligated a double stranded oligonucleotide (the splinkerette) that 1) contains a compatible sticky end, 2) contains a stable hairpin loop, and 3) is unphosphorylated (Figure 2). Two rounds of nested PCR are then performed to amplify the genomic sequence between the transposon insertion and the annealed splinkerette. This is followed by a sequencing reaction with another nested primer. The spPCR reaction remains highly efficient and specific due to the splinkerette design. Since the splinkerette oligonucleotide is not phosphorylated at its 5’ sticky end, only the bottom 3’ recessed strand of the splinkerette sticky end is ligated to the 5’ phosphorylated sticky end of digested genomic DNA. In addition, the PCR primer (‘S1’ in Figure 1) which anneals to the splinkerette only amplifies DNA that has been generated as a result of a successful first strand synthesis. As a result, the PCR reaction occurs preferentially between genomic DNA that has ligated to a splinkerette oligonucleotide. In addition, background products are reduced due to the stable hairpin loop on the splinkerette: 1) it will not ligate to genomic DNA to generate non- specific priming and 2) it reduces end-repair priming (Horn et al., 2007). Since the enzyme does not need to cut within the transgene, any restriction enzyme that produces sticky ends can be used with the appropriate splinkerette oligonucleotide. As such, larger genomic fragments flanking the transgene insertion site can be isolated.

We have adapted spPCR for the mapping of transposable elements (both P-elements and piggyBacs) in Drosophila. The spPCR protocol is simple, efficient, and highly effective. To date, almost every transgene we have attempted to map (n>500) could be mapped by spPCR. Splinkerette PCR could be applied to the mapping of transgenes which proved difficult or impossible using iPCR or plasmid rescue, or for routine mapping of transposable elements in the fly.

Fluorescence-guided Single Sensillum Recording

Fluorescence-guided Single Sensillum Recordings (FgSSR) is a method for performing GFP-targeted SSR in Drosophila.

Full details can be found in Lin and Potter, PLoS One, 2015

 

Traditional single sensillum recordings (SSR) rely on the sizes, shapes, locations and neuronal odor responses (typically to a panel of up to 10 standard odors) to correctly identify target sensilla. We acquired all available OrX-Gal4 lines from the Bloomington Stock Center, and validated which lines, when combined with 10xUAS-IVS-mCD8GFP (on Chromosome 2) or 15xUAS-IVS-mCD8GFP (on Chromosome 3), drove sufficient expression in only a single type of olfactory neuron and were still strong enough to allow identification of sensilla when viewed on a SSR compound microscope setup (see Table below). This significantly shortens the time span needed to identify small sensilla (e.g. ab4-ab10, pb1-3, ai1-3) but also provide a direct method to record from genetically identifiable sensilla.

 

Sensilla (Neuron)

Receptors

Bloomington Stocks for Validated transgenic lines for FgSSRa

Suggested mounting positions

ab1

Or92a, Or42b, Gr21a, Or10a

No needb

Medial

ab2 (A)

Or59b

23897

Medial

ab2 (B)

Or85a

24461

Medial

ab3 (A)

Or22a

9951

Medial

ab3 (B)

Or85b

23911

Medial

ab4 (A)

Or7a

GAL4 Knock-In

Posterior

ab4 (B)

Or56a

23896

Posterior

ab5 (A)

Or82a

23126

Posterior

ab5 (B)

Or47a

9982

Posterior

ab6 (B)

Or49b

9986

Posterior

ab7 (A)

Or98a

23141

Medial

ab7 (B)

Or67c

23905

Medial

ab8 (A)

Or43b

23894

Posterior

ab9 (B)

Or67b

9995

Posterior

ab10 (B?)

Or67a

23904

Medial

at1 (A)

Or67d

GAL4 Knock-In

Posterior or Anterior

at4 (A)

Or47b

9983

Posterior or Anterior

at4 (B)

Or65a

9994

Posterior or Anterior

at4 (C)

Or88a

23138

Posterior or Anterior

ai1 (A)

Or13a

23886

Posterior

ai2 (A)

Or83c

23131

Lateral

ai2 (B)

Or23a

9956

Lateral

ai3 (A)

Or19a

23887

Lateral

ai3 (C)

Or43a

9974

Lateral

pb1c (A)

Or42a

9969

Posterior

pb1c (B)

Or71a

23122

Posterior

pb2c (A)

Or33c

9966

Posterior

pb3c (A)

Or59c

23899

Anterior

pb3c (B)

Or85d

24148

Anterior

a  OrX-Gal4 lines on II were recombined with 10xUAS-IVS-mCD8GFP (Chr II attP40; BS#32186).

  OrX-Gal4 lines on III were recombined with 15xUAS-IVS-mCD8GFP (Chr III attP2; BS#32193).

b ab1 sensilla are the only large basiconic sensilla that contain 4 neurons and thus very easily 

   identified.

c Mounting positions of pb sensilla are less strict since they are more uniformly distributed.

  Homozygous lines (OrX-Gal4, UAS-mCD8GFP/OrX-Gal4, UAS-mCD8GFP) are not recommended in

  maxillary palp ORNs as thinner cuticles cause high fluorescence signals from the

  cell bodies. The thicker cuticle of the antenna greatly reduces visible cell body fluorescence and

  allows for the use of homozygous reporter lines when identifying antenna sensilla. Homozygous

  reporter lines are especially useful when targeting trichoid sensillum.

 

This technique helped clarify the classifcation of intermediate and trichoid sensilla.

There are 2 trichoid sensilla in D. melanogaster (at1, at4), and 3 intermediate sensilla (ai1, ai2, ai3). 



HACK

Bloomington Stock #

Brief Description Genotype
66464 alrm-QF2^G4H y[1] w[*]; Pin[1]/CyO; P{w[+mC]=alrm-QF2.L}3/TM6B, Tb[1]
66465 D42-QF2^G4H y[1] w[*]; PBac{y[+mDint2] w[+mC]=10XQUAS-6XGFP}VK00018, P{w[+mC]=UAS-mtdTomato-3xHA}2; P{w[+mW.hs]=ET-QF2.GB}D42
66466 elav-QF2^G4H P{w[+mW.hs]=ET-QF2.GB}elav[C155-QF2]
66467 GH146-QF2^G4H y[1] w[*]; P{w[+mW.hs]=ET-QF2.GB}GH146/CyO; P{w[+mC]=lacW}mirr[B1-12]/TM6B, Tb[1]
66468 Hml-QF2^G4H y[1] w[*]; P{w[+mC]=Hml-QF2.Delta.L}2; P{w[+mC]=lacW}mirr[B1-12]/TM6B, Tb[1]
66469 Mef2-QF2^G4H y[1] w[*]; Pin[1]/CyO; P{w[+mC]=Mef2-QF2.L}3/TM6B, Tb[1]
66470 NP2222-QF2^G4H y[1] w[*]; P{w[+mW.hs]=ET-QF2.GB}Akap200[NP2222-QF2]/CyO
66471 NP2631-QF2^G4H y[1] w[*]; P{w[+mW.hs]=ET-QF2.GB}NP2631
66472 OK107-QF2^G4H y[1] w[*]; PBac{y[+mDint2] w[+mC]=10XQUAS-6XGFP}VK00018, P{w[+mC]=UAS-mtdTomato-3xHA}2; P{w[+mW.hs]=ET-QF2.GB}ey[OK107-QF2]
66473 OK371-QF2^G4H y[1] w[*]; P{w[+mW.hs]=ET-QF2.GB}VGlut[OK371-QF2]
66474 pebbled-QF2^G4H P{w[+m*]=ET-QF2.GU}peb[QF2]; Pin[1]/CyO
66475 ppk-QF2^G4H y[1] w[*]; PBac{y[+mDint2] w[+mC]=10XQUAS-6XGFP}VK00018, P{w[+mC]=UAS-mtdTomato-3xHA}2; P{w[+mC]=ppk-QF2.L}3/TM6B, Tb[1]
66476 r4-QF2^G4H y[1] w[*]; PBac{y[+mDint2] w[+mC]=10XQUAS-6XGFP}VK00018, P{w[+mC]=UAS-mtdTomato-3xHA}2; P{w[+mC]=r4-QF2.L}3
66477 repo-QF2^G4H y[1] w[*]; Pin[1]/CyO; P{w[+m*]=ET-QF2.GU}repo/TM6B, Tb[1]
66478 tub-QF2^G4H y[1] w[*]; P{w[+mC]=tubP-QF2.L}LL7/TM6B, Tb[1]
66479 GMR57C10-QF2^G4H y[1] w[*]; PBac{y[+mDint2] w[+mC]=10XQUAS-6XGFP}VK00018, P{w[+mC]=UAS-mtdTomato-3xHA}2; P{y[+t7.7] w[+mC]=GMR57C10-QF2.L}attP2
66480 GMR58E02-QF2^G4H y[1] w[*]; PBac{y[+mDint2] w[+mC]=10XQUAS-6XGFP}VK00018, P{w[+mC]=UAS-mtdTomato-3xHA}2; P{y[+t7.7] w[+mC]=GMR58E02-QF2.L}attP2
66481 GMR10A06-QF2^G4H y[1] w[*]; PBac{y[+mDint2] w[+mC]=10XQUAS-6XGFP}VK00018, P{w[+mC]=UAS-mtdTomato-3xHA}2; P{y[+t7.7] w[+mC]=GMR10A06-QF2.L}attP2/TM6B, Tb[1]
66482 GMR20A02-QF2^G4H y[1] w[*]; PBac{y[+mDint2] w[+mC]=10XQUAS-6XGFP}VK00018, P{w[+mC]=UAS-mtdTomato-3xHA}2; P{y[+t7.7] w[+mC]=GMR20A02-QF2.L}attP2
66483 GMR24C12-QF2^G4H y[1] w[*]; PBac{y[+mDint2] w[+mC]=10XQUAS-6XGFP}VK00018, P{w[+mC]=UAS-mtdTomato-3xHA}2; P{y[+t7.7] w[+mC]=GMR24C12-QF2.L}attP2
66484 GMR71G10-QF2^G4H y[1] w[*]; PBac{y[+mDint2] w[+mC]=10XQUAS-6XGFP}VK00018, P{w[+mC]=UAS-mtdTomato-3xHA}2/CyO; P{y[+t7.7] w[+mC]=GMR71G10-QF2.L}attP2
66485 GMR82G02-QF2^G4H y[1] w[*]; PBac{y[+mDint2] w[+mC]=10XQUAS-6XGFP}VK00018, P{w[+mC]=UAS-mtdTomato-3xHA}2; P{y[+t7.7] w[+mC]=GMR82G02-QF2.L}attP2
66486 GMR16A06-QF2^G4H y[1] w[*]; PBac{y[+mDint2] w[+mC]=10XQUAS-6XGFP}VK00018, P{w[+mC]=UAS-mtdTomato-3xHA}2; P{y[+t7.7] w[+mC]=GMR16A06-QF2.L}attP2
66487 QF2^G4H Donor (23F3) y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*]; P{3xP3-RFP=QF2.G4HACK}23F3/CyO
66488 QF2^G4H Donor (25C1) y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*]; P{3xP3-RFP=QF2.G4HACK}25C1/CyO
66489 QF2^G4H Donor (28E4) y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*]; P{3xP3-RFP=QF2.G4HACK}28E4
66490 QF2^G4H Donor (33E4) y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*]; P{3xP3-RFP=QF2.G4HACK}33E4/CyO
66491 QF2^G4H Donor (34C4) y[1] w[*]; P{3xP3-RFP=QF2.G4HACK}34C4; P{w[+mC]=lacW}mirr[B1-12]/TM6B, Tb[1]
66492 QF2^G4H Donor (38C3) y[1] w[*]; P{3xP3-RFP=QF2.G4HACK}38C3; P{w[+mC]=lacW}mirr[B1-12]/TM6B, Tb[1]
66493 QF2^G4H Donor 57C1) y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*]; P{3xP3-RFP=QF2.G4HACK}57C1
66494 QF2^G4H Donor (64B5) y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*]; P{3xP3-RFP=QF2.G4HACK}64B5
66495 QF2^G4H Donor (65E3) y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*]; P{3xP3-RFP=QF2.G4HACK}65E3
66496 QF2^G4H Donor (70F4) y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*]; P{3xP3-RFP=QF2.G4HACK}70F4
66497 QF2^G4H Donor (79A2) y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*]; P{3xP3-RFP=QF2.G4HACK}79A2
66498 QF2^G4H Donor (84C8) y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*]; P{3xP3-RFP=QF2.G4HACK}84C8
66499 QF2^G4H Donor (86E11) y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*]; P{3xP3-RFP=QF2.G4HACK}86E11
66500 QF2^G4H Donor (88B1) y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*]; P{3xP3-RFP=QF2.G4HACK}88B1/TM6B, Tb[1]
66501 QF2^G4H Donor (91A1) y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*]; P{3xP3-RFP=QF2.G4HACK}91A1/TM6B, Tb[1]
66502 QF2^G4H Donor (96D1) y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*]; P{3xP3-RFP=QF2.G4HACK}96D1
66503 QF2^G4H Donor (attP2-PLUS) y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*]; P{3xP3-RFP=QF2.G4HACK.BPGUw}attP2/TM6B, Tb[1]
66504 QF2^G4H Donor (attP2-MINUS) y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*]; P{3xP3-RFP=QF2.G4HACK}attP2