Cell tracking and fate mapping

Question 1

What were some of the issues of previously used dyes such as nile blue and charcoal?

Answer 1

• Easily diffusable
• Short term
• Toxic (cannot study in vivo)

Question 2

What are the advantages of new dyes such as DiI and DiO?

Answer 2

• Fluoresce and allow us to see two things at once
• Bind to the membrane and are non-diffusable
• Permanent
• Non-toxic

Question 3

How is neural fate mapping carried out?

Answer 3

• Cells are labelled with an indelible dye
• 3D coordinates are put in place
• Embryo grows and the positions of the labelled cells are recorded
• Data used to define territories

Question 4

How can chicken/quail chimeras be made?

Answer 4

• Heterotopic grafting

- Quail cells are identifiable by Fulgen staining due to differing amounts of heterochromatin

Question 5

What is the order of cells in the neural crest?

Answer 5

• Cells closer to the head are older

- Cells closer to the tail are younger

Question 6

What can chimeras be used to investigate?

Answer 6

Can inject somite cells into a host at a similar stage to observe where they go and how they react to signals in the environment

Question 7

What was shown in duck/quail chimeras when transplanting part of the neural crest?

Answer 7

transplant of prosencephalic mesencephalic R1 and R2 Neural Crest Cells changes beak shape

Question 8

Name 6 ways of inserting DNA into cells

Answer 8

1. Transformation - DNA in solution
2. Transduction - infect with virus
3. Electroporation - apply charge (DNA is a charged molecule)
4. Sonoporation - using sound
5. Gene gun - bombard with gold particles covered with plasmids
6. Microinjection

Question 9

What 2 things are critical in microinjection?

Answer 9

• The size of the embryo

- Must be performed quickly

Question 10

What is electroporation most useful for?

Answer 10

• Labelling cells at later stages with ubiquitous/cell type specific promoter linked to marker
• Often produces mosaics
• Can look at the timing and location of events, number of progeny and cell migration

Question 11

What is cre recombination?

Answer 11

The permanent integration of a genetic label using cre which is a recombinase derived from P1 bacteriophage

Question 12

How can cre be used to create GFP expression in a desired cell type?

Answer 12

• Create mouse with promotor directing cre expression in a desired cell type
• Mate with mouse with an ubiquitous promoter and a stop codon preventing GFP expression
• Cre-mediated removal of stop codon drives GFP expression in targetted cell type in progeny

Question 13

What 3 things can be used to control GFP system?

Answer 13

1. Light
2. Temperature
3. Drugs