Navigation Links
Mammalian Expression Vectors with Interchangeable Selectable Markers

Site-specific recombinase-based vector system

Lisa Marsh Carsten Carstens

Stratagenes Exchanger system of eukaryotic expression vectors offers unparalleled convenience. The drug-resistance genes of these vectors can be exchanged without subcloning. Through site-specific recombination, hygromycin, puromycin, and neomycin functional cassettes are quickly and efficiently inserted into the pExchange core vectors. Insertion is carried out in vitro by a Cre-mediated recombination of LoxP sites, and recombination products are selected via drug resistance. The components of the Exchanger system include Cre recombinase; one of the six different pExchanger core vectors; linearized insertion modules, which are flanked by LoxP sites and provide hygromycin, puromycin, or neomycin resistance for selection in mammalian cells; the corresponding luciferase core-vector control; and supercompetent Epicurian Coli XL1-Blue cells. The entire site-specific recombination procedure consists of a 30-minute recombination reaction, a 15-minute heat inactivation, and transformation of suitable hosts with the recombination reaction. Desired reaction products are obtained with greater than 90% efficiency.

Stratagenes pCMV-Script and pCMV-Tag mammalian expression vectors1,2,### offer easy cloning and high-level expression; accordingly, they are used in a variety of standard molecular biology applications. These vectors, in addition to containing an expression cassette for inserting the gene of interest into a multiple cloning site, usually include a cassette that contains a selectable marker. However, when it is necessary for the expression vector to carry a different selectable marker, the most common method is to subclone the gene of interest into a different vector, which is very inconvenient. Therefore, it would be ideal to be able to quickly and efficiently exchange drug-resistance elements without subcloning into such vectors.

Cre-Mediated Recombination

To eliminate conventional restriction digestion and ligation subcloning procedures, site-specific recombination is used to manipulate DNA fragments. Site-specific recombinases, such as the P1-derived Cre recombinase, catalyze the exchange of DNA located between specific recognition sequences.3,4,5 For Cre recombinase, the recognition site (LoxP) consists of two inverted 13-bp repeats and an intervening, 8-bp nonpalindromic region.3 Intermolecular and intramolecular recombination between the two sites can occur.

Exchange of Selectable Markers


Stratagenes Exchanger cloning system is based on Cre-mediated, site-specific recombination, which allows fast and efficient insertion of prefabricated, selectable DNA fragments into core expression vectors. The system consists of a pExchange core expression vector, drug-resistance modules and Cre recombinase protein. The pExchange core vectors are based on the pCMV-Script and pCMV-Tag vectors vectors (Figure 1) that contain a LoxP site that serves as the acceptor site for the eukaryotic selectable-marker cassettes and offer a variety of expression options. The hygromycin-, puromycin-, or neomycin-resistance modules (Figure 2) are designed to be inserted into the LoxP site of either core vector. Each cassette is linearized and flanked by LoxP sites. A luciferase-containing core vector control for verifying expression in any cell line is provided along with Epicurian Coli XL-1 Blue supercompetent cells for simplifying transformation. All components are available separately.


The essential process for Cre-mediated insertion of antibiotic-resistance cassettes into the core vectors of the Exchanger system is described in Figure 3. Recombination requires a 30-minute incubation of the core vector, antibiotic-resistance cassette, and Cre recombinase. This reaction is followed by heat inactivation of Cre recombinase at 65C for 15 to 30 minutes. The inactivated recombination reaction is then used to transform host cells, and the transformation mixture is selected on the appropriate antibiotic-containing nutrient plates. After incubation, colonies are picked and screened using standard miniprep procedures, and plasmid DNA can be analyzed by restriction digestion. Successful insertion rates of greater than 90% are achieved routinely.


Functional Testing of the Selectable Cassettes

To test the performance of the eukaryotic selectable markers provided by each cassette, we transfected Chinese hamster ovary (CHO) and NIH3T3 cells with the core vector containing the selectable marker that was introduced by site-specific recombination. Each construct conferred resistance to the respective drug at a frequency equal to or better than comparable vectors with the same drug resistance marker (data not shown).

To confirm the functionality of the CMV expression cassettes of the core vectors, we compared luciferase expression from the pExchange core vectors to luciferase expression from the parental pCMV-Script and pCMV-Tag vectors. In these transient transfections of CHO cells, the presence of the integrated drug-resistance cassettes did not affect the performance of the CMV expression cassette in luciferase expression assays. The observed expression level of the Exchanger system vectors did not differ from their respective parental pCMV-Script and pCMV-Tag vector constructs (data not shown).


Stratagenes Exchanger vector system allows a single expression construct to be used for multiple applications without subcloning the gene of interest. This system features the pExchange core vectors into which, through Cre-mediated site-specific recombination, hygromycin-, puromycin-, or neomycin-resistance selection cassettes can be introduced. Several test experiments have verified that the desired recombinants are generated with greater than 90% efficiency. While eliminating tedious subcloning procedures, the Exchanger vector system provides an easy mechanism for inserting selectable markers.


  1. Hosfield, T. and Lu, Q. (1997) Strategies 10: 116-118.

  2. Abremski, K., Hoess, R., and Sternberg, N. (1983) Cell 32: 1301-1311.

  3. Broach, J.R., Guarascio, V.R., and Jayaram, M. (1982) Cell 29: 227-234.

  4. Landy, A. (1989) Annu. Rev. Biochem. 58: 913-949.



Page: All 1 2 3 4

Related biology technology :

1. New Mammalian Two-Hybrid System Detects Protein-Protein Interactions
2. Highest Transfection Efficiency of an Endotoxin-Sensitive Mammalian Cell Line
3. An Epitope Tagging Vector for Gene Expression in Mammalian Cells
4. Versatile Vectors for Ponasterone A- Inducible Control of Gene Expression in Mammalian Cells
5. New Mammalian Expression Vectors Employ Stable, High-Level Fluorescence Humanized Renilla GFP Reporter
6. A New Lambda Vector for Mammalian Expression
7. Mammalian and Bacterial Expression in One Vector
8. Mammalian Expression Vector for Efficient Cloning of PCR Fragments
9. Low-Toxicity, Lipid-Mediated Transfection of Mammalian Cells
10. Mycoplasma Contamination Reduces the Effect of Lipid-Mediated Transfection of Mammalian Cells
11. Reagents for Up and Down-Regulation of miRNA Activity in Mammalian Cells
Post Your Comments:

(Date:11/27/2015)... 2015 --> ... companion diagnostics is one of the major ... pharmaceutical companies and diagnostic manufacturers working together ... . --> ... global cancer biomarkers market spread across 89 ...
(Date:11/25/2015)... 2015 --> ... - 2020 report analyzes that automating biobanking workflow ... in long-term samples, minimizing manual errors, improving the ... manual errors such as mislabeling or inaccurate sample ... plays a vital role in blood fractionation, DNA ...
(Date:11/25/2015)... , November 25, 2015 ... cat and human plaque and pave the way for more ... problems in cats     --> ... most commonly diagnosed health problems in cats, yet relatively little ... now. Two collaborative studies have been conducted by researchers from ...
(Date:11/25/2015)... CITY , Nov. 25, 2015 /PRNewswire/ - Aeterna ... affirms that its business and prospects remain fundamentally ... , Zoptrex™ (zoptarelin doxorubicin) recently received DSMB recommendation ... to completion following review of the final interim ... Phase 2 Primary Endpoint in men with heavily ...
Breaking Biology Technology:
(Date:11/4/2015)... , November 4, 2015 ... new market report published by Transparency Market Research "Home Security ... Trends and Forecast 2015 - 2022", the global home security ... 30.3 bn by 2022. The market is estimated to ... period from 2015 to 2022. Rising security needs among ...
(Date:10/29/2015)... 2015  Rubicon Genomics, Inc., today announced an ... its DNA library preparation products, including the ThruPLEX ... Plasma-seq kit. ThruPLEX Plasma-seq has been optimized for ... libraries for liquid biopsies--the analysis of cell-free circulating ... in cancer and other conditions. Eurofins Scientific is ...
(Date:10/27/2015)... Germany , October 27, 2015 ... SMI,s Automated Semantic Gaze Mapping technology (ASGM) automatically maps ... SMI,s Eye Tracking Glasses , so that they ... BeGaze. --> Munich, Germany , ... (ASGM) automatically maps data from mobile eye tracking videos ...
Breaking Biology News(10 mins):