The Gateway cloning System, invented and commercialized by Invitrogen since the late 1990s, is a molecular biology method that enables researchers to efficiently transfer DNA-fragments between plasmids using a proprietary set of recombination sequences, the "Gateway att" sites, and two proprietary enzyme mixes, called "LR Clonase", and "BP Clonase". Gateway Cloning Technique allows transfer of DNA fragments between different cloning vectors while maintaining the reading frame. It has effectively replaced the use of restriction endonucleases and ligase

Ligase

In biochemistry, ligase is an enzyme that can catalyse the joining of two large molecules by forming a new chemical bond, usually with accompanying hydrolysis of a small chemical group dependent to one of the larger molecules...

s. Using Gateway, one can clone or subclone

Subcloning

In molecular biology, subcloning is a technique used to move a particular gene of interest from a parent vector to a destination vector in order to further study its functionality....

 DNA segments for functional analysis. The system requires the initial insertion of a DNA fragment into a plasmid with two flanking recombination sequences called “att L 1” and “att L 2”, to develop a “Gateway Entry clone” (special Invitrogen nomenclature).

Large archives of Gateway Entry clones, containing the vast majority of human, mouse and rat ORFs (open reading frame

Open reading frame

In molecular genetics, an open reading frame is a DNA sequence that does not contain a stop codon in a given reading frame.Normally, inserts which interrupt the reading frame of a subsequent region after the start codon cause frameshift mutation of the sequence and dislocate the sequences for stop...

s) have been cloned from human cDNA

Complementary DNA

In genetics, complementary DNA is DNA synthesized from a messenger RNA template in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA polymerase. cDNA is often used to clone eukaryotic genes in prokaryotes...

 libraries or chemically synthesized to support the research community using NIH (National Institutes of Health) funding (e.g., Mammalian Gene Collection, http://mgc.nci.nih.gov/). The availability of these gene cassettes in a standard Gateway cloning plasmid helps researchers quickly transfer these cassettes into plasmids that facilitate the analysis of gene function. Gateway cloning does take more time for initial set-up, and is more expensive than traditional restriction enzyme and ligase-based cloning methods, but it saves time, and offers simpler and highly efficient cloning for down-stream applications.

The technology has been widely adopted by the life science research community especially for applications that require the transfer of thousands of DNA fragments into one type of plasmid (e.g., one containing a CMV promoter for proteins expression in mammalian cells), or for the transfer of one DNA fragment into many different types of plasmids (e.g., for bacterial protein expression, insect expression, and mammalian expression).

Basic Steps

The first step in Gateway cloning is the preparation of a Gateway Entry clone. Entry clones are often made in two steps: 1) “Gateway attB1, and attB2” sequences are added to the 5’, and 3’ end of a gene fragment, respectively, using gene specific PCR primers and PCR-amplification; 2) the PCR amplification products are then mixed with special plasmids called Gateway “Donor vectors” (Invitrogen nomenclature) and the proprietary “BP Clonase” enzyme mix. The enzyme mix catalyzes the recombination and insertion of the att-B-sequence-containing PCR product into the att P recombination sites in the Gateway Donor vector. Once the cassette is part of the target plasmid, it is called an "Entry clone" in the Gateway nomenclature, and recombination sequences are referred to as the Gateway “att L” type.

The gene cassette in the Gateway Entry clone can then be simply and efficiently transferred into any Gateway Destination vector (Invitrogen nomenclature for any Gateway plasmid that contains Gateway “att R” recombination sequences and elements such as promoters and epitope tags, but not ORF

Open reading frame

In molecular genetics, an open reading frame is a DNA sequence that does not contain a stop codon in a given reading frame.Normally, inserts which interrupt the reading frame of a subsequent region after the start codon cause frameshift mutation of the sequence and dislocate the sequences for stop...

s) using the proprietary enzyme mix, “LR Clonase”. Thousands of Gateway Destination plasmids have been made and are freely shared amongst researchers across the world. Gateway Destination vectors are similar to classical expression vectors containing multiple cloning sites, before the insertion of a gene of interest, using restriction enzyme digestion and ligation. Gateway Destination vectors are commercially available from Invitrogen, EMD (Novagen) and Covalys.

Since Gateway cloning uses patented recombination sequences, and proprietary enzyme mixes available only from Invitrogen, the technology does not allow researchers to switch vendors and contributes to the lock-in effect

Vendor lock-in

In economics, vendor lock-in, also known as proprietary lock-in or customer lock-in, makes a customer dependent on a vendor for products and services, unable to use another vendor without substantial switching costs...

of all such patented procedures.

To summarize the different steps involved in Gateway cloning:

• Gateway BP reaction: PCR-product with flanking att B sites (e.g., amplified from cDNA library) + Donor vector containing att P sites + BP clonase => Gateway Entry clone, containing att L sites, flanking gene of interest
  • this step can be replaced by other cloning methods
• Gateway LR reaction: Entry clone containing att L sites + Destination vector containing att R sites, and promoters and tags + LR clonase => Expression clone containing att B sites, flanking gene of interest, ready for gene expression.