Construction of multiple cis-trans vectors linked by 2A polypeptides
Construction of multiple cis-trans vectors linked by 2A polypeptides
This chapter describes methods for expressing multiple proteins from a single readable frame by using 2A polypeptide-linked polycis-trans vectors. When these small sequences are cloned into the intergenic region, they allow efficient production of abundantly unlinked protein products from a single vector by a novel cleavage in the 2A polypeptide sequence. Author: T. Friedman et al, Translated by Wei Qin et al. This experiment is from "Gene Transfer".
Operation method
Generation of 2A-linked polycis-transposon readable frames by recombinant PCR Move Generation of 2A-linked polycis-transposon readable frames by recombinant PCR Material reagents Agarose gel suitable for purification of D N A Dissolve high purity agarose by boiling 2 to 3 m i n I X T A E . Water bath at 55°C until temperature equilibrium. Add 3~5M EB. after gelling, fill the container with IXTAN and remove the comb. Use 1 % agarose gel for fragments larger than 500bp. For smaller products, increase the agarose concentration by 2%. Cloning Vectors There are many different types of commercially available vectors encoding different selectable reading frames, antibiotic resistance genes, and fluorescent proteins. It is important to make sure that all vector systems do not detect 2A peptide-mediated "cleavage" and then consider cloning strategies. D N A Polymerase and Buffer Product length, G/C ratio in the template, and primer Tm affect enzyme selection. We generally use the Advantag^HF 2 PCR kit (Clontech), which produces a consistent 3 kb product with a low mutation rate. Other high-fidelity enzymes (e.g., Phusion High-Fidelity DNA Polymerase, Finnzymes; amplification template, RocheApplied Science), which have been used successfully in our laboratory, are selected depending on the template and primer length used, according to the manual. The selection depends on the template and primer length used, according to the operating manual. D N A Size Markers d N T P Preparation 1 0 mmol/LdN TP, aseptic water solubilization, PCR grade deionized water6 Dispensing Preservation I 80°C Long term (> 2 months) -20°C (< 2 months) EB (1 0 m g /m l) Add Ig EB to IOOml deionized water and stir for several hours until completely dissolved. Store in an opaque bottle at room temperature. 2 5 % bromophenol blue, 0 . 0.25 % Bromophenol Blue, 0.25 % Dimethylbenzene FF, 50 % glycerin dissolved in water) H2O P C R grade P C R primer oligonucleotide P C R grade sterile water preparation working concentration 20u m o l/L , store in portions at -2 0 °C Template D N A PCR with IOOng plasmid DNA or cDNA. 50 X T A E (T r is/A c e ta te /E D T A ) Dissolve 242 g T r i s S 57. Iml glacial acetic acid, IOOml ○ . 5mol/L EDTA (186. Ig EDTA -2 H20 disodium salt, add water to 1L, adjust p H to 8 . 0 ) , diluted with deionized water to ix t a e used. Instrumentation Horizontal gel electrophoresis apparatus for nucleic acid separation, including power supply and appropriate separation combs Use a 3 to 5mm comb to sample the digestive solution. Large sample volumes are used for gel recovery. Therefore, a 7 to 10 mm wide-toothed comb is recommended. PCR Purification or Gel Recovery Kits P C R Instrument Thin-walled PCR tubing UV Lamps method 1. The following reactions were prepared to produce the initial PCR product: Polymerase buffer 5. 0ul and stop codon sequences instead of including the 2A sequence (Fig. 3). For recombinant PCR it is not necessary to change the recovery temperature. The mass of the primer product increases with a corresponding increase in extension time. More than two PCR products can be used for recombinant PCR, provided that the overlapping sequences between the fragments are different and the molar amount of each fragment is the same. If the experiment is difficult to produce recombinant products, recombine the PCR reaction in several stages, using 2 to 3 initial templates per stage, until full-length products are produced. Be aware that increasing the number of PCR cycles increases the probability of error. 7 . Detect the molecular mass of the product as in step 3 and purify the product as in step 4. 8 . Restriction enzyme cleaves the cleavage site in the extension primer and clone the final product into the expression vector. 9. Sequencing is performed using primers specific to the expressed gene or vector of interest. It is important to ensure that the 2A polypeptide sequence is correct, as any changes in amino acid composition will affect cleavage efficiency. reagents Anti-protein or 2A antibody D M E M medium Complete medium and serum-free medium. The complete medium was supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamic acid, 1 mmol/L sodium propionate, 100umol/L MEM non-essential amino acids, 5 mmol/L HEPES, 5.5 x 10-5 units of β-mercaptoethanol, lOOU/ml penicillin and lOOug/ml streptomycin. In addition, 20ug/ml ciprofloxacin Lysate (e.g., Nona Lysate -P 40 or R IP A Lysate Buffer, see H arlo w and L ane 1999) Phosphate Buffer (PB S) Plasmid D N A Transfection reagents Many transfection reagents are available, but we have found that 293T cells can be transfected with T m nsIT-LTl, Tm nSIT-293T (M ims, Madison, Wisconsin), or FuGENE 6 transfection reagent (Roche). If these reagents are not available, calcium phosphate transfection is also possible. Trypsin/Ethylenediaminetetraacetic acid 2 9 3 T is adherent; trypsin ED TA can be used to induce cell detachment from the culture plate to reduce cell clumping Exponential growth 293T cells Instrumentation S D S ^ P A G E Western hybridizer Standard Tissue Culture Instrument, including I O O m m plates This protocol is designed to culture 293T cells in IOOmm tissue culture plates. If different plate sizes are used for cell culture, adjust the cell density and reagent dosage appropriately. METHODS 1. Cells were collected by trypsin digestion 24 h before transfection. a. Remove the adherent cell culture medium (DMEM), rinse with PBS to remove traces of culture medium, and add 3~4m l of trypsin E D T A . b . Incubate at room temperature for 2~3m i n until cells are detached from the culture plate. Gently resuspend the cells by transferring them to a conical tube, wash the plate with IOml complete medium, transfer the rinsing medium to the tube, and neutralize the trypsin. c. lOOOr/min Centrifuge for lOmin. Remove supernatant, resuspend precipitate in 5m l of medium, and count cells. Inoculate cells into IOOmm tissue culture plates at a concentration of 2 X IO6 cells/plate with IOml of medium. Overnight at 37°C to allow the cells to adhere to the wall. 2. Select the reagents to transfect the cells according to the instructions. Incubate the cells at 37 °C for more than 48 h using 100ug vector/plate. 3. Collect the cells by trypsin digestion as in step 1. 4- Detection of cleavage, lysis of cells, S D S - P A G E electrophoresis to separate proteins, and W e s t e r n hybridization. Standard procedures for blocking and probe selection are appropriate for the target protein. If antibodies to the study protein are available W e s t e r n hybridization is effective and can be used to test the efficiency of cleavage. Antibodies to 2A peptides are not commercially available, but can be prepared on their own. Depending on the protein molecular mass, they can identify the protein of interest to distinguish it from cleaved and non-cleaved material. Proteins expressed in cells transfected with multiple cis-transfector vectors can be compared with cells transfected with a separate protein pellet. It is important to realize that 2A labeling causes small changes in the molecular mass of proteins For more product details, please visit Aladdin Scientific website.
Template D N A (IOOng) I .Oul
can prevent mycoplasma contamination. All reagents and media are commercially available.