producing transgenic organisms

Question 1

why make transgenic organisms?

Answer 1

there are many reasons other than pure research why making transgenic organisms is important:

1 – manufacture of proteins useful in medicine (e.g. human insulin, growth hormone)

2 – introduction of novel traits into commercially valuable organisms (crop/farm animal improvement)

3 – xenografting (organs useful for human transplantation)

4 – biotechnology – manufacture of useful proteins (e.g. restriction endonucleases, Taq)

Question 2

What is cloning?

Answer 2

there is a lot of confusion about terms – cloning is a term we use for producing many copies of a piece of DNA but is also the term for producing identical animals/plants

Question 3

cloning of animals does not necessarily involve …

Answer 3

…genetic modification

Question 4

prior to the advent of recombinant DNA technology insulin for diabetics was ‘manufactured’ by collection of …

Answer 4

… pig or cattle pancreas tissue from abattoirs

Question 5

the insulin was then collected from …

Answer 5

… the pancreas and purified.

Question 6

Problems with using cow and pig insulin/pancreas tissue?

Answer 6

problems - not human insulin (efficiency?)

possible infection risk (e.g. human growth hormone - CJD)

Question 7

insulin A and B chains are transformed separately as …

Answer 7

fusion proteins

Question 8

insulin A and B chains are transformed separately as fusion proteins

the transformed bacteria are grown separately in …

Answer 8

… fermenters and the insulin/fusion protein collected

Question 9

Describe the process of making human insulin?

Answer 9

Two genes cloned into plasmids.

Plasmid 1, (left) has a promoter, antibiotic res gene, a subunits
Second plasmid, anti biotic res gene, promoter, b subunits

Plasmids grown in cultures of e.coli in fermenters.
Extract cells from the culture at various points – extract and purify
Treat with cyanogen bromide to remove beta galactokinase
Purify and mix a and b chains of both plasmids to form functional units.
Joined by disulphide bond.