Winter storms can wreak havoc on air travel plans. Much like inserting genes into expression vectors, you may set out for one destination and find yourself (or your luggage) in another. Airlines will help you and your belongings reach your city or town eventually, usually after one too many hours in an unfamiliar airport. Genes, on the other hand, need a good dose of luck to arrive at that perfect spot on the circular plasmid or linear strand—unless you’re using the newest line of products, which provide maximum reliability through sophisticated genetic engineering.
Gregor Mendel didn’t have access to the latest products and technology when he conducted his famous genetic experiments on peas. Nevertheless, his system of cross-pollination hinted that phenotypic traits were not just an accident. By the early 1970s, Hamilton O. Smith began pioneering techniques that used restriction enzymes as tools for genetic studies. He shared the 1978 Nobel Prize in Medicine and Physiology for his efforts. Soon after, researchers used restriction enzymes to insert the insulin gene into a plasmid vector, thus giving birth to the biotechnology industry.
Times have changed, to put it mildly. With the human genome completely sequenced, researchers are driving full speed ahead to understand most every gene. “However, transfection efficiency [of plasmids] varies widely between cell types, and automatization costs and efforts are considerable,” according to a paper published in the June 2006 issue of the Journal of Virology. (M Hillgenberg et al., “High-Efficiency System for the Construction of Adenovirus Vectors and Its Application to the Generation of Representative Adenovirus-Based cDNA Expression Libraries,” Journal of Virology, 80: 5435–5450, 2006.) “Therefore, a variety of virus-derived vector systems have been developed for improved cDNA transduction, expression, and screening in mammalian cells.”
While restriction enzymes remain popular for constructing both bacterial and viral vectors, many researchers are embracing methods that depend on recombination. In this process, the gene of interest is placed into an entry vector between two recombination target sites, such as att or loxP. Upon entering the cell and lining up with similar sites displayed by another vector, the enzyme recombinase assists in inserting the gene of interest into the other vector. Companies offer various types of gene-inserting systems with different target sites and recombinase, such as clonase and cre.
Some products are designed for inserting multiple genes into one vector. Whether for protein-protein interaction or expression analysis studies, the latest systems also maximize your time by saving you from performing multiple plasmid transfections. You can choose from kits that allow inserting only two or three genes. Or, for more flexibility, purchase the kit that allows up to four genes. If you have spare change, you may consider purchasing pre-made entry vectors with a clone of your choice. Along with the gene(s) of interest, you can also insert tags that are transcribed with the gene. Once translated, the protein displays the tags, which can be used for straightforward identification and purification with readily available antibodies.
Take a look at the list below to get an idea of the market’s offerings. You’ll soon realize that they’re the only way to fly.
Enhanced Protein Detection, Tracking and Characterization
Our pCMV-3Tag vectors enhance signal strength in all of your protein characterization studies by adding three copies of either the FLAG® or c-Myc tags to your protein. The FLAG and c-Myc tags are small — 8 (DYKDDDDK) and 10 (EQKLISEEDL) amino acid residues, respectively — highly immuno-reactive, do not interfere with the function of your protein, and are easily detected with our anti-FLAG or anti-c-Myc antibodies. Triple epitope-tagged proteins are detected with up to 60-fold higher sensitivity than proteins with a single tag.
