How would you genetically engineer a gene construct to produce human antithrombin in goat’s milk?
The correct answer and explanation is:
To genetically engineer a gene construct to produce human antithrombin in goat’s milk, the following steps would be undertaken:
- Gene Cloning: The gene encoding human antithrombin (AT) needs to be isolated from human DNA or synthesized. This gene is then cloned into a suitable expression vector (such as a plasmid). The vector will carry the necessary regulatory elements to ensure gene expression in the goat mammary gland. These elements include a strong promoter (like the β-casein promoter) that is active in the goat’s mammary tissue during lactation.
- Insertion of the Gene into the Expression Vector: The gene for human antithrombin is inserted into the expression vector using restriction enzymes and ligation. The vector will also include a polyadenylation signal, which is essential for the proper termination of the transcription process in the mammary gland cells.
- Transfection of Goat Embryos or Somatic Cells: The next step is the introduction of the gene construct into the goat genome. This can be done using two primary methods:
- Somatic Cell Nuclear Transfer (SCNT): This involves transferring the nucleus of a somatic cell (a cell from the goat with the inserted gene) into an enucleated goat egg cell. The embryo is then implanted into a surrogate mother for development.
- Microinjection into Zygotes: The gene construct is injected directly into fertilized goat eggs, which are then implanted into a surrogate mother.
- Selection and Screening: After the goat is born, it is necessary to screen its DNA to confirm that the human antithrombin gene has been successfully integrated into the genome. The animals are also monitored for the expression of the gene in their milk. A combination of PCR, Southern blotting, and Western blotting can be used to confirm both gene presence and protein expression.
- Milk Collection and Protein Purification: Once the transgenic goats are lactating, their milk is collected, and the human antithrombin protein is purified. Techniques such as affinity chromatography, using a specific antibody to bind to human antithrombin, would be employed to isolate the protein.
The use of goats for biopharming (producing proteins in animal milk) is advantageous because it allows for large-scale production of complex proteins that are difficult to produce in bacterial or yeast systems. In this case, human antithrombin, which is used to treat individuals with antithrombin deficiency, can be produced in significant quantities.