Practise matching gene-transfer enzymes to their roles, evaluating plasmid vectors and sequencing the steps that create and deliver recombinant DNA.
Traditional techniques for genetically modifying organisms use three enzymes:
- restriction endonuclease
- reverse transcriptase
- DNA ligase.
For example, these enzymes have been used to produce genetically modified (transgenic) pigs containing the GFP gene coding for green fluorescent protein, originally sourced from jellyfish.
Outline how these three enzymes could be used in genetically engineering a transgenic pig containing the GFP gene.
A new technique that aims to cause a deletion in a gene uses an enzyme called Cas9 nuclease. It is injected into zygotes along with an RNA sequence (the guide RNA) that is complementary to a target gene. The Cas9 nuclease causes a deletion in the target gene in the zygotes, preventing the expression of that gene.
The toxicity and efficiency of the new technique was tested on four groups of pig zygotes. These pig zygotes were produced by IVF using:
- ova from a female non-transgenic pig.
- sperm from a male transgenic pig whose somatic (body) cells contained one copy of the GFP gene per cell.
The pig zygotes in three groups were injected with different concentrations of Cas9 nuclease and guide RNA targeted at the GFP gene.
The fourth group of pig zygotes (control group) was not injected with Cas9 nuclease and guide RNA.
1 restriction endonuclease cuts, vector / plasmid ;
A restriction enzyme
2 reverse transcriptase to make cDNA using mRNA ;
3 DNA ligase joins sugar phosphate backbone (between gene and vector) or DNA ligase forms phosphodiester bonds (between gene and vector) ;