In vivo gene cloning

Can you remember... What we mean by in vitro and in vivo?

So far.... You have found out about obtaining fragments of DNA by: Using reverse transcriptase Using restriction endonucleases

You can then make copies of these genes using which in vitro technique? PCR In vivo cloning can also be used to copy genes

Steps in in vivo cloning In vitro to start with Stage 1 Stage 2 Stage 3 Stage 4

Stage 1 – obtaining the DNA for the gene we want to clone, we looked at this last week Stage 2 – is insertion of the DNA into a vector A vector is a carrier and is required in order to transfer the extracted gene into a chosen bacterial cell. An example of a vector is a plasmid i.e. a circular piece of DNA found in a bacterium.

How do we insert the gene into the plasmid How do we insert the gene into the plasmid? (refs p 6 booklet p249-251 A2 book) The plasmid is treated with the same restriction endonuclease enzyme as the extracted DNA. This means it has complementary sticky ends to the extracted DNA so they can be joined together by the enzyme DNA ligase.

Stage 3 – inserting the plasmid into bacterial cells Bacteria and plasmids are mixed in a medium containing calcium ions. This makes the bacterial cell wall more permeable to plasmids, which pass into the cytoplasm of the bacteria. Bacteria which contain the recombinant plasmid are said to be transformed

Try this exercise with your pieces of human and plasmid DNA (You will need to remember the recognition site for ECoR1. ) Treat the human DNA with Eco R1 restriction endonuclease enzyme ( ) Use your Eco R1 restriction enzyme i.e to cut the plasmid and produce sticky ends complementary to your human gene. Use your DNA ligase i.e. To join the sticky ends of the plasmid with those of the human gene You have now made a recombinant plasmid!!

Adenoviruses and liposomes are other examples of vectors, apart from plasmids

Steps in in vivo cloning In vitro to start with Stage 1 Stage 2 Stage 3 Firstly, we need to identify which bacteria contain the recombinant plasmids Stage 4

Identifying Transformed Host Cells These are cells that have taken up the plasmid Use antibiotic resistance Gene for antibiotic resistance is found in some plasmids, so insert required gene into them Grow bacteria on medium containing antibiotic Only those bacteria that have taken up the plasmid will survive!

Antibiotic resistance markers FOREIGN GENE eg insulin MARKER GENE eg antibiotic resistance

How can the transformed bacteria be identified? By culturing the bacteria on an agar plate which contains the antibiotic. Untransformed bacteria (no resistance) Transformed bacteria (contain the resistance gene) NO GROWTH GROWTH

Now make sure you have completed all the qs on page 6

How can we identify bacteria which have taken up both the gene and the plasmid? (page 7) We use GENE MARKERS. These identify both the presence of the plasmid AND the presence of the required gene. 3 types of gene marker: Antibiotic resistance Fluorescence An enzyme

Recombinant plasmid produced: Use plasmids with 2 resistance genes (getting complicated and clever now!) Will the tetracycline resistance gene still function? Ampicillin resistance gene Human gene inserted into tetracycline resistance gene Recombinant plasmid produced:

But.... Sometimes the original plasmid rejoins its sticky ends to itself without inserting the foreign gene

So..we need to ensure we only select bacteria with the recombinant plasmid In this example – we first grow the bacteria on a plate containing ampillicin Only bacteria with plasmids containing this resistance gene will grow Now grow the surviving bacteria on the second antibiotic – usually Tetracycline What will happen? This is pointless so what to do?

Replica Plating Take small samples of bacteria surviving the first antibiotic Grow on 2nd antibiotic Note positions of dead colonies These are the ones containing the required gene

Fluorescent Markers Insert gene for fluorescence (from a fish) into plasmid. Insert required gene into the middle of it and put into bacteria Grow bacteria Bacteria that have taken up the gene and the plasmid will NOT fluoresce

Finally...Stage 4 Growth / cloning (in vivo) The transformed bacteria, which have now been identified, are now grown on a large scale in a fermenter. They will secrete the gene product (e.g. insulin) into the medium they are growing in and it can then be extracted and purified.

Now test yourself... Complete the summary questions on page 253 of the text book Answer the exam question on page 25 and 26 of your booklet.