1. In vivo gene cloning

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

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

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

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

7. 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.

8. How do we insert the gene into the plasmid
How do we insert the gene into the plasmid? (refs p 6 booklet p 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.

9. 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

10. 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!!

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

12. 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

13. 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!

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

15. 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

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

17. 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

18. 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:

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

20. 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?

21. 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

22. 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

23. 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.

24. 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.