KNOCK ‐ OUT IN CELL LINES VIA TALEN TECHNOLOGY
II.9.2. A SSEMBLY OF AKR1B15 TALEN PAIRS
The cloning of the TALEN constructs aimed for the knock‐out of either one or both of the AKR1B15 isoforms (AKR1B15.1, AKR1B15.2, or AKR1B15‐E2) was performed with the Golden Gate TALEN and TAL Effector Kit 2.0 (Addgene). This kit consists of plasmids enabling the position specific assembly of any custom TALEN pair and is based on the technique developed by Cermak et al. [224]. The assembly of the final TALEN pairs covered in principle two cloning steps and was mainly carried out according to the protocol used by the Voytas lab (Golden_Gate_TALEN_assembly_v5.doc) with slightly modifications. The procedure with all alterations is described in the following and illustrated in Figure II‐4.
Before starting the cloning, all plasmids necessary for the assembly of AKR1B15 TALEN constructs [Table II‐19] were purified from mini‐scale E. coli cultures carrying the respective plasmid using the NucleoSpin Plasmid (Macherey Nagel) kit. the insertion of the RVDs into the vector backbones. The digestion reactions were separated on a 1 % agarose gel and the pFUS‐A/B vector backbone (2500 bp) was purified from the gel using the Wizard SV Gel and PCR Clean‐Up System (Promega). For the first cloning step, 20 μl reactions (called Golden Gate Reaction #1) were set up. The reactions comprised of either 150 ng of the purified pFUS‐A vector backbone and 150 ng of each of the first ten module vectors (position 1‐10) or 150 ng of the purified pFUS‐B(n‐11) vector backbone and 150 ng of each of the remaining module vectors (position 11‐(n‐1)) as well as 10 U of the restriction enzyme BsaI (NEB) and 400 U of the T4 DNA Ligase (NEB) in 1x T4 DNA Ligase Buffer. BsaI is a restriction enzyme that cuts outside of the recognition sites (one bp downstream of the 3’ base and five bp upstream of the 5’ base of the recognition site) and, therefore, allows for the generation of variable overhangs for ligation reactions and the elimination of restriction sites after ligation. The overhangs of BsaI restriction sites in the module vectors are designed in a way that enables the sequential assembly of RVDs. The concerted digestion (at 37 °C) and ligation (at 16 °C) of plasmids was performed in a RoboCycler 96 (Stratagene) using the program listed in Table II‐20.
Table II‐20: PCR program used for the Golden Gate Reaction #1.
Subsequently, in order to get rid of all linear and unligated DNA fragments, the reactions were treated with 1 μl Plasmid‐Safe ATP‐dependent DNase (Epicentre Biotechnologies) and 0.5 μl ATP (25 mM) at 37 °C for 60 min. Afterwards, the DNase treatment was stopped by an incubation step at 70 °C for 30 min. These reactions were then transformed into chemically competent E. coli DH5α cells and streaked out together with X‐Gal/IPTG on LB‐agar‐Spec(50) plates allowing for blue‐white screening (see II.1.2).
The next day, a colony screen using the pCR8_F1 forward (# 2698) and pCR8_R1 reverse (# 2698) primer pair was performed with white colonies from the plate. Plasmids of potential positive clones were amplified in overnight cultures, isolated, and sequenced using the primers pCR8_F1 (# 2698) and pCR8_R1 (# 2698).
For the second cloning step, resulting in the final TALEN constructs, 20 μl reactions (called Golden Gate Reaction #2) were set up. These reactions included 150 ng of each intermediate pFUS‐A (RVD 1‐10) and pFUS‐B (RVD 11‐(n‐1)) vector, 150 ng of the respective pLR vector, which encodes for the final RVD, and 75 ng of the pcGoldyTALEN destination vector. The successive digestion of RVD encoding plasmids as well as the final target vector and the ligation reactions of the resulting linear DNA fragments were catalyzed by the addition of 10 U of BsmBI (NEB) and 400 U of the T4 DNA Ligase (NEB) in 1x T4 DNA Ligase Buffer.
Just as BsaI, BsmBI is a restriction enzyme that cuts outside the recognition sites (one bp downstream of the 3’ base and five bp upstream of the 5’ base of the recognition site). The restriction sites are again designed in a way that leads to variable overhangs allowing for the correct assembly of the TALEN fragments and eliminating the restriction site. The reactions were performed in a RoboCycler 96 (Stratagene) using the program listed in Table II‐21. chemically competent E. coli DH5α cells and streaked out together with X‐Gal/IPTG on LB‐agar‐Amp(100) plates allowing for blue‐white screening (see II.1.2).
The next day, a colony screen using the TAL_F1 forward (# 2700) and TAL_R2 reverse (# 2701) primer pair was performed with white colonies from the plate. Plasmids of potential positive clones were amplified in overnight cultures, isolated, and sequenced using the forward primers TAL_F1 (# 2700) or SeqTALEN_5‐1 (# 2702) as well as the reverse primer TAL_R2 (# 2701).
Figure II‐4: Schematic illustration of the cloning procedure for AKR1B15 TALENs.
The assembly of TALENs was performed in two cloning steps (Golden Gate Reaction #1 and Golden Gate Reaction #2) via concerted restriction digestions (using the BsaI and BsmBI enzymes, respectively) and ligation reactions (using the T4 DNA ligase).
HD, His‐Asp di‐residue; NG, Asn‐Gly di‐residue; NH, Asn‐His di‐residue, NI, Asn‐Ile di‐residue; NLS, nuclear localization signal; pFUS‐A/B, intermediate target vectors; pHD, pNG, pNH, and pNI, module vectors; pcGoldyTALEN, final target vector; RVD, repeat‐variable di‐residue.
II.9.3. C
LONING OF SELECTION MARKER FORTALEN
TRANSFECTIONSelection markers are a good tool for the identification of successfully transfected cells. Here, different selection markers under the control of a CMV promoter (CMV‐AcGFP, CMV‐DsRed, and CMV‐ΔLNGFR) [Table II‐22] were cloned the into pcGoldyTALEN target and effector vectors via the DraIII restriction site using the In‐Fusion HD Cloning Plus (Clontech) kit.
Table II‐22: Primers and templates used for the amplification of selection marker.
selection marker primers for cloning template CMV‐AcGFP # 2945 + # 2946 pAcGFP‐N1 CMV‐DsRed # 2945 + # 2946 pCMV DsRed‐Express2 CMV‐ΔLNGFR # 2943 + # 2944 pMACS‐ΔLNGFR
The cloning was largely carried out according to the manufacturer’s instructions, however, taking only half of the volumes recommended. The procedure is described briefly in the following.
Following the manufacturer’s recommendations, the selection markers were amplified in 25 μl reactions containing 90 ng plasmid template, primers in a final concentration of 200 nM each, as well as the 2x CloneAmp HiFi PCR‐Mix (Clontech) [Table II‐22]. In order to enable the In‐Fusion recombination reactions, 5 μg of the respective pcGoldyTALEN plasmids were linearized in 1x CutSmart Buffer with 20 U of DraIII‐HF (NEB) at 37 °C overnight. Both, PCR products as well as linearized plasmids, were purified via the NucleoSpin Gel and PCR Clean‐Up Kit (Macherey‐Nagel) before 35‐40 ng linearized plasmid and 40 ng PCR product were applied in 5 μl In‐Fusion cloning reactions containing also 1 μl of the 5x In‐Fusion HD Enzyme Premix. Differing from the recommendations of the manufacturer, these cloning reactions were incubated at 50 °C for 30 min before 1.2 μl were transformed into 50 μl Stellar Competent Cells (Clontech) and plated on LB‐agar‐Amp(100) plates for selection [II.1.2].