Mammalian system Protein production and nucleofection: Nucleofection of mammalian cells transient protein production stable protein production Tech note coming soon Nucleofection of insect cells Optimized Protocols coming soon Protein production and nucleofection Transient nucleofection of mammalian cells: Fast protein expression within a few days Transfection in serum-free suspension cultures Higher efficiencies than with competitor methods Suited for validation, optimization and characterization experiments Substantial cost and time saving! Transient nucleofection of mammalian cells Example of change in the expression of chosen protein:
Natural cloning[ edit ] Cloning is a natural form of reproduction that has allowed life forms to spread for hundreds of millions of years. It is the reproduction method used by plantsfungiand bacteriaand is also the way that clonal colonies reproduce themselves.
Molecular cloning Molecular cloning refers to the process of making multiple molecules. Cloning is commonly used to amplify DNA fragments containing whole genesbut it can also be used to amplify any DNA sequence such as promotersnon-coding sequences and randomly fragmented DNA.
It is used in a wide array of biological experiments and practical applications ranging from genetic fingerprinting to large scale protein production. Occasionally, the term cloning is misleadingly used to refer to the identification of the chromosomal location of a gene associated with a particular phenotype of interest, such as in positional cloning.
In practice, localization of the gene to a chromosome or genomic region does not necessarily enable one to isolate or amplify the relevant genomic sequence.
To amplify any DNA sequence in a living organism, that sequence must be linked to an origin of replicationwhich is a sequence of DNA capable of directing the propagation of itself and any linked sequence.
However, a number of other features are needed, and a variety of specialised cloning vectors small piece of DNA into which a foreign DNA fragment can be inserted exist that allow protein productionaffinity taggingsingle stranded RNA or DNA production and a host of other molecular biology tools.
Subsequently, a ligation procedure is used where the amplified fragment is inserted into a vector piece of DNA. The vector which is frequently circular is linearised using restriction enzymesand incubated with the fragment of interest under appropriate conditions with an enzyme called DNA ligase.
Following ligation the vector with the insert of interest is transfected into cells. A number of alternative techniques are available, such as chemical sensitivation of cells, electroporationoptical injection and biolistics.
Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low efficiency, there is a need to identify the cells that have been successfully transfected with the vector construct containing the desired insertion sequence in the required orientation.
Modern cloning vectors include selectable antibiotic resistance markers, which allow only cells in which the vector has been transfected, to grow. Nevertheless, these selection steps do not absolutely guarantee that the DNA insert is present in the cells obtained.
Further investigation of the resulting colonies must be required to confirm that cloning was successful.
Cloning unicellular organisms[ edit ] Cloning cell-line colonies using cloning rings Cloning a cell means to derive a population of cells from a single cell. In the case of unicellular organisms such as bacteria and yeast, this process is remarkably simple and essentially only requires the inoculation of the appropriate medium.
However, in the case of cell cultures from multi-cellular organisms, cell cloning is an arduous task as these cells will not readily grow in standard media.
A useful tissue culture technique used to clone distinct lineages of cell lines involves the use of cloning rings cylinders.Gateway® technology allows highly efficient transfer of the gene of interest into any of a number of expression vectors from a single entry clone.
The variety of expression choices available makes the Gateway® system of recombination cloning ideal for protein expression studies. Lewis et al.
() identified and cloned Mecp2 from a rat brain cDNA library. The deduced amino acid protein has a molecular mass of 53 kD and is rich in basic amino acids and potential phosphorylation sites. The Gateway recombination cloning system is designed for highly efficient transfer of your DNA into any Gateway expression vector from a single entry clone.
The extremely wide variety of Gateway cloning–compatible expression vectors available makes the Gateway system of recombination cloning ideal for protein expression studies.
Abstract. Inducible production of proteins from cloned genes in attheheels.com is widely used, economical, and effective. However, common practices can result in unintended induction, inadvertently generating cultures that give poor or variable yields in protein production.
These include having an effect on whether or not the protein can fold correctly, whether the protein is active, the way in which the protein is recognized by the immune system, and the Gene-ch9-cpp 10/8/06 Page Production of Proteins from Cloned Genes Obtain cloned gene Place gene in a suitable vector with a strong promoter.
Nitrogen Metabolism. Nitrogen is a very important constituent of cellular components. Alkaloids, amides, amino acids, proteins, DNA, RNA, enzymes, vitamins, hormones and many other cellular compounds contain nitrogen as one of the elements.