Fast and efficient PCR cloning with pcmv-script PCR cloning kit
Tanya Hosfield Kerstien Padgett Tim Sanchez Quinn
Stratagene Cloning Systems, Inc.
Stratagene has expanded the applications of the PCR-Script cloning strategy by introducing a new vector for high-efficiency cloning of PCR fragments and expression in mammalian cells. The pcmv-script PCR cloning kit uses a PCR cloning method that features a one-hour ligation and doesnt require adding extra bases to the primers. In this method, the PCR products are incubated with Srf I-digested pCMV-Script vector, Srf I and T4 DNA ligase. Ligating insert and vector in the presence of T4 DNA ligase and Srf I enzyme reduces the background of self-ligated vector. We have tested the pCMV-Script vector with two different reporter genes and have confirmed high-level expression of the gene products in mammalian cells.
Cloning PCR-generated fragments using PCR-Script cloning kits is a popular method,1 proven to be efficient, easy and superior to other available methods.2 In this cloning method, PCR products are incubated with the SrfI-digested pPCR-Script vector, Srf I restriction enzyme and T4 DNA ligase. The Srf I enzyme recognizes the rare sequence 5-GCCCGGGC-3. By including Srf I enzyme in the ligation reaction, the PCR-Script cloning method maintains high concentrations of digested vector DNA and allows the use of nonphosphorylated, unmodified PCR products. The ligation efficiency of blunt-ended DNA fragments is increased by the simultaneous, opposing reactivity of the Srf I enzyme and the T4 DNA ligase on nonrecombinant vector DNA.
In many instances, researchers prefer to characterize a eukaryotic gene in mammalian cells in order to analyze cor rect folding and posttranslational modifications of the gene product. Such posttranslational modifications are often required for the activity of eukaryotic proteins. To make the PCR-Script cloning strategy available to researchers interested in expressing their genes in eukaryotic cells, Stratagene has created the pCMV-Script PCR cloning kit, a mammalian version of the PCR-Script cloning system.
The pcmv-script PCR cloning kit features the pCMV-Script vector (figure 1), a mammalian expression vector that uses the cytomegalovirus (CMV) promoter for constitutive expression in a wide variety of cell lines. The pCMV-Script vector shares many essential characteristics with the pPCR-Script vector: capacity to clone inserts generated with any PCR enzyme, high efficiency and 1-hour ligation. However, the pCMV-Script vector has many distinguishing features. The powerful CMV promoter and the SV40 polyadenylation site allow high-level constitutive expression in a wide variety of cell lines. In addition, the neomycin-resistance gene is under control of the prokaryotic b-lactamase promoter to provide kanamycin resistance in bacteria as well as the SV40 early promoter to provide G418 resistance in mammalian cells.
The pCMV-Script PCR cloning kit provides reagents for 25 reactions. Each kit contains predigested pCMV-Script vector, cloned Pfu DNA polymerase for polishing,3 Srf I restriction enzyme, T4 DNA ligase and Epicurian Coli XL10-Gold ultracompetent cells. The kit also includes a control PCR insert and the necessary cloning and polishing buffers.
The cloning efficiency of the pCMV-Scrip t PCR cloning kit was verified using a test gene. To reduce the number of potential mutations that result during PCR amplification and to generate blunt-ended DNA fragments, we chose Pfu DNA polymerase for PCR amplification of the ampicillin-resistance gene. The pCMV-Script vector was digested with Srf I restriction endonuclease and ligated to the PCR-amplified ampicillin-resistance gene in the presence of Srf I to eliminate the background of recircularized vector. This mixture was transformed into XL10-Gold cells, and 50 l of the 500 l transformation mix was plated on LB agar plates containing kanamycin. In order to determine the percentage of colonies that contained the insert, 100 of the resulting 250 colonies were streaked onto an LB agar plate with ampicillin. Of the recovered transformants, 73% contained the ampicillin-resistance-gene insert.
The pCMV-Script vector was tested in both transient and stable transfection assays. The green fluorescent protein gene (GFP) and the firefly luciferase gene were used for transient transfection assays. The GFP gene was cloned into the pCMV-Script vector, and the resulting construct was transfected using standard lipid transfection methods into Chinese hamster ovary (CHO) cells. The expression of the GFP gene product was detected by Western blot analysis (data not shown).
Similarly, the luciferase gene was cloned into the pCMV-Script vector and used to transfect CHO cells. In order to compare expression levels of genes inserted into the pCMV-Script vector with expression levels in other available vector products, the luciferase gene was also inserted into the pcDNA 3.1 vector. Each construct and appropriate controls were used to transfect CHO cells. Forty-eight hours following transfection, CHO cells were harvested, luciferase activity was quantitated (figure 2) and the protein of the correct molecular weight was detected by Western blot analysis (data not shown). Luciferase activity and expression from the pCMV-Script vector were equivalent to that from the pcDNA 3.1 vector.
The pCMV-Script vector was stably transfected into CHO cells using Stratagenes Mammalian Transfection Kit. In separate experiments, the pCMV-Script vector and the pCMV-Script vector containing the luciferase gene were transfected into CHO cells, and G418 selection was used to obtain stable cell lines. Luciferase expression from stable cells containing the pCMV-Script vector with the luciferase gene insert was compared to cells containing the pCMV-Script vector with no insert. Detected luciferase activity was 1000-fold over background, measured by a standard luciferase assay (data not shown).
Stratagenes new pCMV-Script cloning kit offers high-efficiency cloning of blunt-ended PCR fragments for expression in mammalian cells. The pCMV-Script vector features (1) the CMV promoter for expression in a variety of cell lines, (2) the kanamycin- and neomycin-resistance gene for selection in either prokaryotic or eukaryotic systems and (3) ligation of insert to the pCMV-Script vector in the presence T4 DNA ligase and Srf I to eliminate background of recircularized vector. The pCMV-Script vector can be used to clone inserts that have been generated by any PCR enzyme. We have tested this vector and demonstrated high-level expression in mammalian cells.
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