STEP 1. Creation of SNAP- and HaloTag expression constructs lTIMING up to several weeks
p CRITICAL STEP The engineering of constructs is a sustainable challenge for CIDs and
extensive testing and optimization of the constructs for every single application is required, which is the most time consuming part of CID experiments
1| Create expression constructs with SNAP- and HaloTag dimerizing domains
(i) Compile DNA sequences of expression constructs containing SNAP- and HaloTag domain:
“effector translocation” approach:
anchor unit: anchoring motif, SNAP- or HaloTag, fluorophore
effector unit: POI, SNAP- or HaloTag, fluorophore “mutual dimerization” approach:
effector unit 1: POI1, SNAP- or HaloTag, fluorophore effector unit 2: POI2, SNAP- or HaloTag, fluorophore Design of anchor unit
-if available use a ready-to-use plasmid from anchor unit library (see Fig 7 and Table 2), if desired available for your anchor of interest (Golgi, endosomes, lysosomes, plasma membrane, mitochondrium, actin skeleton as well as the subcellular compartments outside and inside the nucleus)
-if other organelles / subcellular compartments are targeted: design new anchor units by inserting a SNAP- resp. HaloTag dimerizing domain and fluorescent reporter into a functional plasmid containing the desired anchoring motif, while retaining the context of the targeting motif as good as possible (N- versus C-terminal-fusion, linkers between domain and targeting motif)
Design of effector unit
-design plasmid containing the POI, SNAP- resp. HaloTag dimerizing domain and fluorescent reporter, if available take construct containing the POI, insert SNAP- resp. HaloTag domain, keep original context of POI as good as possible possible (N- versus C-terminal-fusion, linkers between domain and targeting motif)
(ii) Amplify DNA fragments using PCR primers that contain 18 – 25 bases annealing to the 5´ resp. 3´end of the gene fragment, add 15 bases homologous to sequences flanking the insertion site in the backbone to facility In-Fusion recombination. Alternatively,
amplify DNA fragments with conventional cloning using restriction digest and T4 DNA Ligase
(iii) Set up 50 µl reaction for each DNA fragment to be amplified by PCR
5x Phusion HF or GC buffer 10 µl
Template DNA (10 ng/µl) 1 µl
Results
Annealing 45-72°C 20 sec
Elongation 72°C 15 sec per 1 kb
30 cycles
Final elongation 72°C 10 min
Hold 4°C
(v) Digest 2 µg of plasmid DNA with amount of Units of restriction enzymes and buffer (according to manufacturerʼs protocol) for 1 h at the appropriate temperature in a total volume of 20 µl.
(vi) Analyze undigested PCR and digested backbone by agarose gel electrophoresis, gel purify with GenElute (Gel Extraction Kit Sigma Aldrich)
(vii) Set up In-Fusion reaction in 10 µl according to manufacturerʼs protocol: x µl digested backbone (10 – 200 ng)* x µl DNA fragment (50 – 200 ng)** 0.5 µl In-fusion reaction mix fill up to 10 µl water
*<0.5 kb: 10-50 ng, 0.5 to 10 kb: 50-100 ng, >10 kb: 50-200 ng **<10 kb: 50-100 ng, >10 kb: 50-200 ng
Incubate the reaction mix for 15 min at 50°C.
(viii) Use 5 µl of In-Fusion reaction to transform CaCl2-competent E.coli cells (XL1 blue) and select for growth by plating on LB agar plates containing the appropriate antibiotics for selection (depending on the backbone plasmid). Incubate ON at 37°C.
PAUSE POINT: Plate can be stored at 4°C for several weeks, In-Fusion reaction mix can be stored indefinitively at -20°C.
(ix) Pick several clones, isolate DNA using GenElute Plasmid Miniprep Kit (Sigma Aldrich) and verify clones by analytical restriction digestion and sequencing.
à Perform TEST EXPERIMENT 1 to verify localization and translocation of anchor and effector unit.
STEP 2. Creation of cells co-expressing SNAP- and HaloTag expression constructs lTIMING ~ 24 h
p CRITICAL STEP Find the optimal ratio of expression levels of the anchor unit to effector
unit, at which the effector unit can be completely integrated in the induced dimer, but at which its expression level still sufficient high to allow induction of its correlated cellular effect.
2| Seed and transfect HeLa or HEK293 cells with SNAP- or HaloTag expression constructs,
for microscopic analysis follow STEP 2A, for analysis in cell lysates follow STEP 2B.
(A) Preparing cells for analysis in cell lysates
(i) Day 1. Trypsinize growing HEK293 or HeLa cells using trypsin-EDTA and count the cells using a hemocytometer
(ii) Resuspend 8 Î106 HEK293 resp. 1.5 Î106 HeLa cells in a total volume of 2 ml DMEM medium
(iii) Manually pipette cell suspension in each well of a 6-well plate (Falcon)
p CRITICAL STEP The number of plated cells is crucial that plated cells be attached,
forming a monolayer and transfection efficiency is high (most efficient if 60-80% confluent at day of transfection)
(ii) Day 2. Transient transfection of SNAP- and HaloTag expression constructs with JetPei according to manufacturers protocol: 0.5 µg total DNA / well in 24-well plate, 2 µg total DNA / well in 6-well plate, use ratio of amount of DNA of both constructs according to desired expression levels
(B) Preparing cells for microscopic analysis
Results
p CRITICAL STEP The number of plated cells is crucial and the plated cells should be
attached and to form a monolayer. Transfection efficiency is most efficient if cells are 60- 80% confluent at day of transfection.
(ii) Day 2. Transient transfection of SNAP- and HaloTag expression constructs with JetPei according to manufacturerʼs protocol: 0.5 µg total DNA per well in 24-well plate, use ratio of amount of DNA of both constructs according to desired expression levels
à Perform TEST EXPERIMENT 2 to verify localization and translocation of anchor and effector unit
Alternatively, use another cell line with suitable cell culture medium at appropriate temperature and suitable amount of cells.
STEP 3. HaXS treatment of cells co-expressing SNAP- and Halo expression constructs 3| Induce dimerization by incubating cells with HaXS dimerizer either following protocol for cell
lysates (STEP 3A) or protocol for live cells (STEP 3B)
(A) Perform experiment in cell lysates lTIMING ~ 60 min
(i) Day 3. Dilute HaXS8 dimerizer (from 10 mM stocks in DMSO) in pre-warmed cell culture medium to an end concentration of 5 µM
(ii) Vortex medium with HaXS dimerizer
(iii) Add 300 µl medium per well in 24-well and 900 µl per well in 6-well medium containing HaXS to the cells expressing the anchor and effector unit
(iv) Swirl plate to mix
(v) After suitable time of HaXS incubation at 37°C perform analysis or cleavage (STEP 4A)
Alternatively, directly pipette DMSO stock solution of HaXS into wells with transfected cells. (B) Perform experiment in living cells lTIMING ~ 2- 3 h
(i) Day 3. Transfer coverslips with cells co-expressing SNAP- and HaloTag expression constructs to pre-warmed Ludin chamber (Life Imaging Services), closed configuration (ii) Carefully add 300 µl pre-warmed imaging medium (phenol red free DMEM cell culture
medium) in Ludin chamber
(iii) Attach tube and syringe at Ludin chamber and transfer Ludin chamber into Ludin chamber holder in microscope in the temperature incubation box
(iv) Start imaging by live cell microscopy
(v) Carefully add 300 µl of phenol red free cell culture medium containing 2x HaXS dimerizer concentration
(vi) Image dimerization until complete, take picture every 1 to 15 sec depending on effect under investigation
p CRITICAL STEP While working with a new cell line, the different cell penetration behavior
of HaXS can have an effect on the optimal concentration and thus it is important to determine the optimal concentration at which HaXS induce most efficient dimerization (perform analog to TEST EXPERIMENT 3, but with concentration gradient (0.5 µM to 10 µM) instead of time course for HaXS incubation)
STEP 4. Cleavage of MeNV-HaXS-induced dimers
4| Induce cleavage of MeNV-HaXS dimers by illuminating cells or subcellular region of cells
either following protocol for cell lysates (STEP 4 A) or protocol for live cells (STEP 4B)
(A) Perform experiment in cell lysates lTIMING ~ 30 min
(i) Day 3. Remove cell culture medium, wash cells twice with 1x PBS and submerge cells with 1x PBS
(ii) Illuminate cells with UV lamp (Blak-Ray B-100A high intensity UV lamp; 100 Watt, 365 nm) for 5 min, distance of lamp around 5 cm, keep cells on ice to prevent warming of cells
Results
(iii) Illuminate cells for appropriate time with:
-355 nm FRAP laser (diode laser, 50 mW, XY scanning excitation laser) -405 nm laser (laser strength as medium intensity) (UV diode)
p CRITICAL STEP To exclude unbeneficial effects exerted by UV light during cleavage
process, perform control experiment with light-insensitive dimerizer HaXS8 in parallel.
p CRITICAL STEP Illumination time depends on laser strength, fluorescent lamp strength and
the size of the illuminated region (ROI resp. field of view).