Cloning in Bacteria and Eukaryotes
Cloning in bacteria
Bacteria can be classified into (i) gram negative bacteria e.g. Escherichia coli. Pseudomonas, Rhizobium and (ii) gram positive bacteria e.g. Bacillus subtilis and Streptomycetes sp. Ordinarily plasmids can not maintain themselves in stable form in both classes of bacteria and, therefore, plasmid vector for two classes usually differ. While for gram negative bacteria, plasmids, phages and cosmids are known as vectors, for gram positive bacteria, only plasmids are known to do this job.
Cloning in eukaryotes
Since chromosomes found in the nucleus in eukaryotes are separated from the rest of cell through nuclear membrane, and since many of the genes are split genes with exons and introns, genetic engineering with eukaryotes requires new methods and tools. When eukaryotic genes are cloned in prokaryotes, the split genes can not be correctly expressed, because prokaryotes do not have the equipment necessary for splicing out the RNA transcribed from the introns of a gene.
Among eukaryotes, DNA cloning has been done in yeast, mouse and to some extent even in some higher plant species. In yeast, a plasmid called 2μ DNA (63bp) is found, which is an appropriate cloning vehicle. An efficient transformation method is also available, which involves protoplast production and PEG (polyethylene glycol) directed introduction of DNA. In animal cells like mouse cells, special animal viruses were used as cloning vehicles. Simian virus 40 (SV40) is one such virus, in which globin gene could be integrated. This gene integrated in SV40 could be transcribed and translated in mouse kidney cells.