Cell Culture Techniques

Question 1

How are cell line cultures produced?

Answer 1

1. Isolated from cancerous tissues (e.g. HeLa cells)
2. Immortalisation of primary cultures:
   a) Spontaneously from prolonged culture: multiple ill-defined mutations transformed phenotype
   b) Through genetic manipulation: artificial transformation of healthy primary cells

Production through genetic manipulation
• To generate cell lines we target processes that regulate cellular growth and ageing
• As cells divide over time, telomeres shorten, and eventually cell division stops → Apoptosis (p53, pRb)

Question 2

How can we inhibit the function of tumour suppressor proteins, or introduce telomerase in order to alter a cell’s capability for its finite number of divisions?

Answer 2

• Taking advantage of viral ‘oncoproteins and transfecting the cells with telomere gene

Question 3

What is the viral oncogene and cellular targets for SV40 and HPV respectively?

Question 4

What does SV40 T-antigen interact with?

What does this increase?

Answer 4

p53 and pRb

This can cause increased growth without loss of function of these proteins

Question 5

What. does HPV E6 and E7 target?

Answer 5

• HPV’s E6 targets p53 for degradation, and E7 binds to pRb inactivating it

Question 6

What type of phenotype do cell lines made using E6/E7?

Answer 6

• Cell lines made using E6/ E7 oncoproteins are believed to maintain a differentiated phenotype

Question 7

What happens to the telomerase gene?

Answer 7

The telomerase gene can also be introduced into a target primary cell.

Question 8

What do some cells need for immortalisation?

Answer 8

• Some cells need both introduction of the telomerase gene and inactivation of the pRb/p53 for “immortalisation”

Question 9

What does E6/E7 and telomerase transformations result in?

Answer 9

• E6/ E7 and telomerase transformations are believed to result in cell lines with a differentiated phenotype