Cloning vehicle

Bolivar, F., Rodriguez, R. L., Greene, P. J., et al., 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2 95-113. A paper describing one of the early plasmid cloning systems. 

FIGURE 3.13 Insertion of a foreign DNA sequence into a plasmid cloning vehicle, using restriction endonucleases and ligase. 

Another technique is to engineer genes to place a polyhistidine "tag" at the C terminus of a protein chain. Commercial cloning vehicles and kits are available for this purpose.124 The protein produced when the engineered gene is expressed can be captured by the affinity of the polyhistidine tag for Cu2+, Ni2+, Co2+, or Zn2+ held in chelated form on an affinity column.124-127... 

A major goal in recombinant DNA technology is the production of useful foreign proteins by bacteria, yeast, or other cultured cells. Protein synthesis depends upon both transcription and translation of the cloned genes and may also involve secretion of proteins from the host cells. The first step, transcription, is controlled to a major extent by the structures of promoters and other control elements in the DNA (Chapter 28). Since eukaryotic promoters often function poorly in bacteria, it is customary to put the cloned gene under the control of a strong bacterial or viral X promoter. The latter include the X promoter PL (Fig. 28-8) and the lac (Fig. 28-2) and trp promoters of E. coli. These are all available in cloning vehicles. 

Physical methods of gene transfer. Genes can often be transferred without the use of a cloning vehicle. This is especially important for certain plant cells, such as those of cereal grains, for which transfer of genes via the Ti plasmid has been difficult.167 If DNA, which may be in a plasmid, is coprecipitated with calcium phosphate, it can often be taken up directly either by animal cells or by plant protoplasts.168 169 Polycations also facilitate DNA uptake cationic liposomes seem to be especially effective.170 In the widely used electroporation technique a short electrical pulse of a few hundred volts / cm is applied to create transient pores in the plasma membrane through which the DNA can enter a cell.111 8,171 175 Chromosomes can be transferred by cell fusion and either... 

The ideal plasmid cloning vehicle has the following properties ... 

A good cloning vehicle must generally have a number of features ... 

Other features of the phage which add to its usefulness as a cloning vehicle are ... 

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