8.4.5 Culture of Transformed Host Cells

• Polymerase chain reaction (PCR) is a common molecular biology technique used in most applications of gene technology
  • It is described as the in vitro method of DNA amplification

• Gene cloning can also be carried out in vivo, using bacteria
  • Bacteria are the most common host cells for this method as they increase in numbers rapidly and are relatively easy to culture

In vivo gene cloning

• There are several steps involved in the process
• A DNA fragment is isolated
  • The desired gene(s) is obtained by one of three methods
    • Extraction using restriction endonucleases
    • The conversion of mRNA to cDNA using reverse transcriptase
    • Artificial synthesis in a "gene machine"

  • Promoter and terminator regions are added to the fragments of DNA to ensure replication

• The DNA fragments are inserted into vectors
  • Restriction endonucleases and ligase enzymes are used to insert the fragments of DNA into the vector
  • Plasmids are commonly used vectors

• The vectors are transported into bacterial host cells
  • The cells containing the modified plasmids are described as transformed host cells

• Bacteria multiply in number
  • Under the optimum conditions, the bacteria rapidly increase in numbers

• Marker genes are used to identify the successfully transformed bacteria
  • Only a small fraction of the bacteria will have taken up the plasmid containing the desired gene
  • Those that have taken up the plasmid can be identified via markers
  • Markers – genes that code for identifiable substances that can be tracked (eg. GFP – green fluorescent protein which fluoresces under UV light or GUS – a β-glucuronidase enzyme which transforms colourless or non-fluorescent substrates into products that are coloured or fluorescent)
  • Those that have not taken up the desired gene are destroyed

• The remaining bacteria are cultured
  • Every time a bacterium divides the desired gene is cloned

The steps involved in the process of in vivo gene cloning using bacteria as the host cells

• Recombinant DNA can be used to produce recombinant proteins (RP)
• Bacteria have been genetically engineered for the production of human protein insulin to treat diabetes

An overview of the steps taken to genetically engineer an organism (in this case bacteria are being genetically engineered to produce human insulin)