The chimera is then introduced into its host (e.g.,a bacterium) by various methods. Vectors carrying the genes must be incorporated into the living cells so that they can be expressed or replicated. The cells receiving the vector is called the host cell and once the vector is successfully incorporated into the host cell, the host cell is said to be “transformed”.
Illustration of cloning in bacteria
Foreign DNA cannot be readily sent across the membrane, following are few methods.
Heat shock: The chimera plasmids are placed in a solution containing cold calcium chloride and normal host bacteria. On heating suddenly to 42°C for 2-5 minutes the host bacterial membranes become permeable to plasmid chimeras, which pass into the cell.
Electroporation: The host cells are subjected to a high voltage pulse which temporarily disrupts the membrane and allows the vector to enter the cell.
Viruses: Since viruses have mechanism to infect susceptible cell and replicate themselves, a genetically engineered virus can deliver desired DNA sequence into the target host cell.
Gene gun: Gold particles coated with foreign DNA segments are fired into the host cell.
Microinjection: A cell in held in place with a pipette under a microscope and foreign DNA is injected directly into the nucleus using fine needle.
Liposome: Vectors can be enclosed in a liposome, which are small membrane bound vesicles. The liposomes fuse with the cell membrane (or nuclear membrane) and deliver the DNA into the cytoplasm/nucleus.
Selection of transformed cells
A pUC18 plasmid containing gene (lacZ’) coding for galactosidase activity is inserted with a foreign DNA. The plasmid also codes for ampicillin resistance. Due to the insertion, the gene gets interrupted and the bacterium transformed with this plasmid lacks galactosidase activity. Bacteria lacking this plasmid as well as those transformed by the chimeric plasmid lack galactosidase activity. When grown on medium containing a chromogenic substrate, bacteria containing chimeric plasmid produce colourless colonies.
Bacteria containing plasmid without the insert produce blue colonies and the bacteria not transformed by plasmid also produce colourless colonies. If ampicillin is also incorporated in the medium, bacteria not transformed with plasmid do not produce colonies. Thus, on this medium the colourless colonies indicate bacteria that have received chimeric plasmid.
Other methods to detect successful transfer of DNA include DNA hybridization and PCR.
Gene mapping
It involves determining the locations of genes within specific chromosomes. It is a critical step in the understanding of genetic diseases. There are two types of gene mapping, genetic mapping and physical mapping. Genetic mapping is used to determine the relative position of genes within a chromosome. This is measured by whether or not two genes are "linked". If both genes are inherited together they are considered linked. By determining which genes are linked, the relative positions of genes can be worked out. Physical mapping involves determining the exact position of a specific gene within a chromosome. There are multiple techniques for accomplishing this, including somatic cell hybridization and Fluorescent In Situ Hybridization (FISH).
Next: Continuation of genetic engineering part 5: Application of genetic engineering
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