Cell Culture Techniques

Question 1

Name 3 techniques used to isolate cells from blood.

Answer 1

1. Density centrifugation
2. FACS
3. Immunopurification

Question 2

Describe the disadvantages of primary cells.

Answer 2

• Poor growth characteristics
• Inter-patient variation
• Molecular manipulation difficult
• Phenotypic instability

Question 3

Describe 2 ways that cell lines can be produced from primary cultures.

Answer 3

1. Spontaneous - from tumours or prolonged culture
2. Genetic manipulation

Question 4

Name the viral genes from HPV that can be used to modify the activity of p53 and Rb?

Answer 4

• E6 - targets p53 for degradation
• E7 - binds to and inhibits retinoblastoma (Rb)

Question 5

Describe the roles of p53, Rb and telomerase.

Answer 5

• p53 - binds to unprotected end of chromosome once telomeres are depleted
• Rb - inhibits DNA synthesis and arrests cells in G1
• Telomerase - prevents the shortening of telomeres

Question 6

Name the 4 steps in the derivation of a cell line.

Answer 6

1. Gene for selection e.g. ampicillin resistance gene expressed on a plasmid along with growth promoting gene - e.g. E6/E7
2. Transfection
3. Selection pressure added - e.g. antibiotics such as ampicillin, gentomycin
4. Colonies selected

Question 7

Describe 5 methods of transfection.

Answer 7

d

Question 8

Explain the disadvantages of cell lines.

Answer 8

• Rapidly dividing cells often lose differentiated function
• Thus in making lines growth and function are at odds

Question 9

Define cell line.

Answer 9

A cell culture developed from a single cell with a uniform genetic make up.

Question 10

Describe the advantages of cell lines.

Answer 10

• Conditions are as they would be in vivo
• Cell to cell communication is re-established