Construction of MultiLabel vectors with a single expression cassette
1| This step can be performed using option A, B or C depending on the available template. Use option A, if a suitable expression cassette with a promoter, the gene of interest, and a polyA signal already exists. Standard primers for pcDNA (Invitrogen) or pEGFP type (Clontech) vector are described in this paper (Fig. 2A). Use option B, if a protein with an N-terminal fluorescent protein tag is needed (Fig. 2B). Use option C for the restriction enzyme free insertion of a coding region at any place in the MultiLabel vector (Fig. 2C).
A. First option: This strategy inserts an existing expression cassette into MultiLabel vectors by conventional cloning. An existing expression cassette of a pcDNA (Invitrogen) or pEGFP-type (Clontech) vector is amplified by PCR using primers containing AscI and PacI restriction enzyme sites, and then cloned into a AscI- / PacI- cut MultiLabel vector (Fig. 2A). This strategy does not work, if your fragment contains an AscI or PacI site (in this situation ligate 3 fragments, use SLIC or a PCR-based method as described in option 1C).
See the attached document for details on how to do this.
"Procedure and Troubleshooting.doc":http://www.nature.com/protocolexchange/system/uploads/1864/original/Procedure_and_Troubleshooting.doc?1312282214
B. Second option: This option describes the insertion of a gene of interest downstream of the coding region of a fluorescent protein by conventional cloning. MultiLabel vectors contain two SapI (LguI) restriction sites which generate non cohesive ends that can be used to exchange the linker fragment with your gene of interest (Fig. 2B). Make sure the SapI recognition sequence is not present in the genes you are cloning.
See the attached document for details on how to do this.
"Procedure and Troubleshooting.doc":http://www.nature.com/protocolexchange/system/uploads/1864/original/Procedure_and_Troubleshooting.doc?1312282214
C. Third option: Seamless cloning using restriction free methods can be used to insert DNA sequences at any place of MultiLabel vectors. Add a 15–20 bases 5′-tail to the primers containing DNA sequences that are homologous to the sequences flanking the site of insertion into the MultiLabel vector. The described primers bind to the 3’ end of the CMV promoter (RF-CMV-for) and to the5’ end of the poly A signal (RF-PolyA-back, Fig 2C). This method has two PCR steps. In the first PCR the gene of interest is amplified and this PCR fragment replaces in the second step the linker of the MultiLabel vector.
See the attached document for details on how to do this.
"Procedure and Troubleshooting.doc":http://www.nature.com/protocolexchange/system/uploads/1864/original/Procedure_and_Troubleshooting.doc?1312282214
CRITICAL STEPS
Steps 1Ai, 1Bi, 1Ci, and 1Civ It is critical to use a high fidelity proof-reading polymerase with a low error rate. We use Phusion high-fidelity DNA polymerase in 2× HF buffer, but the use of other polymerases is possible. The use of the wax beads reduces non-specific amplification during the PCR. Other hot start methods than wax beads are applicable but more expensive.
Steps 1Aiv, 1Biv, and 1Ciii DpnI removes methylated parental DNA and hemimethylated hybrids of one parental and one PCR synthesized strand. Otherwise methylated parental DNA and hemimethylated hybrids would give rise to high background after transformation.
2| Use 0.5 μl of ligation or restriction free reaction to transform competent E. coli cells (TOP10 or DH10 for acceptor plasmids or BW23474 for donor plasmids) and select on LB agar plates containing the appropriate antibiotic for selection (depending on the vector of choice; see Table 1). Incubate overnight at 37°C.
▲ CRITICAL STEP The correct bacterial strain has to be chosen for transforming cloning products. Donor plasmids and derivatives contain a conditional origin of replication derived from R6Kγ and have to be propagated in cell strains expressing the pir gene such as BW23474. Acceptor plasmids and derivatives can be propagated in common laboratory bacterial strains (for example, TOP10 or DH10β).
TROUBLESHOOTING
3| Pick 2-10 white colonies (see Supplementary Fig. 2A) and inoculate small scale cultures in LB media containing the appropriate antibiotic. The isolated plasmids should then be analyzed by restriction mapping using restriction enzymes.
▲ CRITICAL STEP Phusion polymerase has a low error rate. Nonetheless, it is advisable to verify the inserted gene by sequencing.
Creating multigene expression plasmids
4| Set up plasmid assembly reactions for 3 MultiLabel vectors in a 10 μl volume.
ddH2O: -- 2 μl
10× Cre reaction buffer: -- 1 μl
Acceptor plasmid (1μg): -- 2 μl
Donor plasmid 1 (1μg): -- 2 μl
Donor plasmid 2 (1μg): -- 2 μl
Cre recombinase: -- 1 μl
Incubate for 1 hour at 37°C.
▲ CRITICAL STEP Cre recombinase is a very sensitive enzyme; therefore DNA should be purified with an anion exchange kit.
One or two donor plasmids can be routinely assembled with one acceptor plasmid. The third and fourth donor plasmids should be inserted in a sequential manner with triple-assembled and four-assembled multigene plasmid, respectively.
5| Use 0.5 μl of the Cre recombinase reaction to transform competent E. coli cells (for example, TOP10 or DH10β). Plate the cells on LB agar plates containing appropriate combination of antibiotics (Supplementary Fig. 2B, Table 1). Incubate overnight at 30°C.
6| Pick 2-10 colonies to grow small scale cultures for plasmid isolation in LB media containing the corresponding antibiotics. The isolated plasmids should then be analyzed by restriction mapping using appropriate restriction enzymes.
➨ TROUBLESHOOTING
■ PAUSE POINT Plasmids can be stored at 4°C.
Generation of stable cell lines
Linearization of the multigene plasmid
Several acceptor vectors contain a recognition site for the homing endonuclease I-SceI (Supplementary Figure 1). If one of your genes of interest contains a I-SceI restriction site, the BstZ17I restriction enzyme can be used to linearize the multigene vector. The BstZ17I restriction site is localized on the border between Kanamycin and FRT regions and will not interfere with mammalian expression.
7| Set up preparative restriction digestion reaction in 50 μl volume each:
MultiLabel multigene vector (50 μg) -- x μl
10× digestion buffer (Tango) -- 5 μl
I-SceI restriction enzyme -- 1 μl
ddH20 -- till 50 μl
Incubate for 16h at 37°C.
8| Run an analytic agarose gel to check if the multigene vector is linearized completely. Purify the linearized multigene plasmid using the QIAprep PCR purification kit or phenol-chloroform extraction followed by ethanol precipitation.
▲ CRITICAL STEP It is very important that the multigene plasmid is completely linearized prior transfection into the mammalian cells.
9| Measure the concentration of the linearized multigene plasmid.
Preparation of mammalian cells for transfection in 10cm tissue culture plate
Note that this is our lab standard cell culture protocol for transfection of HEK293 and PAE cells. The protocol may need some adaptations or other cell types.
10| Plate HEK293 or PAE cells 6-9 hours prior transfection in 10cm dishes. Cells should be 30-40% confluent prior to transfection.
▲ CRITICAL STEP From now on, all steps should be performed in a sterile hood.
11| Mix 5 μg linearized multigene plasmid with 7.5 μl FuGENE HD transfection reagent in 250 μl serum-free medium (Opti-MEM I). Incubate for 20 min at room temperature before adding to the plated cells.
▲ CRITICAL STEP In addition to FuGENE HD, there are several other transfection reagents which can be used (for example, PEI or calcium phosphate). The choice depends on the used cell type.
Selection of stable transfectants
12| Cells are passed into 2-4 10 cm plates 40 h after transfection in different dilutions (1:2, 1:5, and 1:20). Add selection drug one day after replating (Table 1). Replace medium once or twice per week, always add drug. Colonies are visible within 14-21 days and should be picked (clone rings with grease for PAE cells or filter cloning discs (Sigma) for HEK293 cells). Transfer colonies to 1cm wells and grow in presence of selection drug. Pass into 3.5 or 6cm plates and freeze aliquots.