8.19 Transfer of Recombinant DNA into Other Cells

• Sections of DNA can be transferred from one organism's DNA to that of another, creating recombinant DNA
• In order to create recombinant DNA, desired genes, such as the gene for human insulin, need to be transferred into new types of cell; there are several different ways of doing this, e.g.
  • Vectors, e.g.
    • DNA plasmids
    • Viruses
    • Liposomes
  • Gene guns
  • Microinjection
• Once a section of DNA has been transferred into a new cell it needs to be incorporated into the cell's genetic material in order to be transcribed and translated; the success rate for transferred genes entering the nucleus of a eukaryotic cell is currently very low
  • The problem of transferred genes not reaching the nucleus is a barrier to the success of gene therapy
    • Gene therapy is a medical technique that involves inserting non-mutated genes into the cells of individuals with genetic disorders

Plasmid vectors

• Plasmids are small, circular rings of double-stranded DNA that can be found in some bacterial cells, as well as some other cell types
• To insert the desired gene into the circular DNA of a plasmid, it is cut open using a restriction endonuclease
• DNA ligase is then used to join the desired gene to the plasmid DNA; this creates a recombinant plasmid
• The recombinant plasmid DNA can then be transferred into other cells, usually bacteria, by a process called transformation
  • Only a small proportion of bacteria will become transformed and therefore marker genes such as antibiotic resistance are used to identify them
    • Scientists can modify or design bacterial plasmids so that they contain one or more marker genes, e.g. genes for antibiotic resistance
• Transformation can occur by
  • Bathing the plasmids and bacteria in an ice-cold calcium chloride solution and then briefly incubating at 40°C; this makes the bacterial outer membrane permeable
  • Electroporation; the bacteria are given a small electrical shock, making the membrane porous

Plasmid vectors can be used to transform bacterial cells

Viral vectors

• Viruses reproduce by inserting their DNA into the cells of other organisms, making them ideal vectors for the process of creating transformed cells
• Viruses exist that infect animal cells, plant cells, and bacterial cells, so viral vectors can be used to transform many cell types
• Viruses used as vectors need to be non-harmful to the cells being transformed
• Viruses are useful for ensuring that transferred DNA reaches the nucleus of cells, but despite being harmless they can sometimes initiate an immune response
• Viruses can be used as vectors in the process of gene therapy, which can be used to treat genetic diseases such as cystic fibrosis in humans

Viral vectors can be used to insert genes into human cells

Liposome vectors

• Liposomes are small, spherical vesicles, surrounded by a phospholipid bilayer 
• Liposomes can be used as vectors because they can fuse with the cell surface membranes of host cells
• These vesicles can also be used in gene therapy to carry non-mutated genes into host cells 

Liposomes can enter cells by fusing with the cell surface membrane. Note that you do not need to know the term endosymbiosis here

Gene guns

• The fragments of DNA containing the desired gene can be used to coat tiny pellets of gold or tungsten which are then fired at high speed into the cells that are to be transformed
• Cells that avoid damage are able to incorporate the new DNA into their genome

Microinjection

• A fine glass micropipette is used to transfer DNA into a cell
• This is a common laboratory technique for transferring genetic material into animal cells for the creation of transgenic animals