International A Level Biology / Gene technology
Getting recombinant DNA into cells revision notes
Edexcel International A Level Biology (YBI11), chapter 25: Gene technology, specification point 8.19. Free lesson.
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Recombinant DNA must enter a living cell so the cell can copy it or make its protein. Different methods suit different cells, and marker genes help identify the cells that took up the vector.
How bacteria take up plasmids
Bacteria and plasmids are mixed in ice-cold calcium chloride. Calcium ions help the negatively charged DNA reach the bacterial cell surface. A brief heat shock to about 42 °C makes the membrane more permeable, so some plasmids enter. The bacteria are then put back on ice and grown in warm conditions. Only a small fraction take up a plasmid. A cell that has taken up new DNA is called transformed.
How Agrobacterium puts genes into plants
Agrobacterium tumefaciens is a soil bacterium that naturally transfers some of its DNA into plant cells. Its DNA comes from the Ti plasmid (tumour-inducing plasmid) and can cause crown-gall tumours. To use it as a vector, the tumour genes are removed and the wanted gene is inserted. The recombinant plasmid is returned to the bacterium, which infects plant cells. Part of the Ti plasmid joins a plant chromosome. Tissue culture can then grow the cells into whole plants, with the new gene in every cell and passed on in seeds. This method works less well in some plants, including many…
Which method suits each cell?
The method depends on the type of cell. Viral vectors are altered so they deliver a gene without causing disease. A gene gun fires DNA-coated gold or tungsten pellets into plant cells. Microinjection uses a fine glass needle to put DNA into the nucleus of a fertilised egg; the embryo is then placed in a surrogate mother. Liposomes are phospholipid spheres containing DNA. They fuse with the phospholipid cell surface membrane and release the DNA inside. Viral vector: animal cells, for example in gene therapy. Gene gun: plant cells, often cereals. Microinjection: fertilised…
How marker genes identify transformed cells
A marker gene is put into the same vector as the wanted gene. If a cell shows the marker, it has taken up the vector. With an antibiotic-resistance marker, bacteria are grown on agar containing the antibiotic. Resistant cells survive and form colonies; the others are killed. With GFP (green fluorescent protein), ultraviolet light makes transformed cells glow green. Other cells do not glow, but remain alive. Some vectors close up without the wanted gene, so a further check is needed to confirm that the gene itself is present. Each colony grows from one cell, so every cell in that…
Choosing and describing a method
Match the method to the cell named in the question. Restriction endonucleases cut the wanted gene out of its original DNA and open a vector. They do not cut the recipient cell's DNA to let the gene in. The gene enters the cell by a delivery method, such as microinjection or a viral vector. A gene gun suits a wheat plant. Microinjection suits a fertilised goat egg.