W2L2 Fri Reverse genetics - technical approaches

Question 1

Retina as neural development model

Answer 1

-simple structure with layer of cells, highly conserved
-a progenitor cell develop into many different cell type. Need high level of coordination to ensure correct number connected at the correct place

Question 2

Multipotency

Answer 2

• The potential/ limitation of a cell to differentiate into other cell type

Question 3

Cre multi-colour cell labelling process

Answer 3

-In the presence of Cre recombinase (conditional), different fluorescent proteins are made. multiple construct
-if there are multiple constructs (e.g. 3), you can get a whole range of different final colours, if Cre recombinase is not 100%
efficient
-This whole construct can be targeted to specific tissues using a tissue- specific “conditional” promoter to confine Cre recombinase

Question 4

Inducible chemical or heats chock

Answer 4

Can be chemically or temperature inducible (using Cre recombinase -oestrogen receptor and adding tamoxifen or heat shock promoter driving Cre recombinase)

Question 5

Use of Cre multicolor cell labelling

Answer 5

Individual cells - cell tracing
Clonal analysis - find all the offspring from one progenitor / stem cell
-look at Developmental and regenerative growth strategies

Question 6

Gene networks and Cre

Answer 6

-Combine transgenesis (fluorescent reporter) to “watch” gene expression with live imaging.
-This cannot be examined in species, where we have to rely on post-mortem analysis of fixed non-living tissue / cells
-Lineage describes gene networks in each progenitor over time: Allows us to determine combination and sequence of genes

Question 7

Using gene loss of fuction and Cre

Answer 7

-Combine transgenesis to mark all cells that activate the promoter with morpholino knockdown
Studying the role of genes
Necessity: Gene knockdown (MO) or knockout (mutant):
Alternate fate of cells (transgenesis to track cells):
Quantify morphology, location and markers (transgenic or immunomarkers) for cell type or cell death

Question 8

Gene gain of function and CRE

Answer 8

-Sufficiency of a gene (reverse genetics): Gal4 / UAS

Question 9

Cell autonomy

Answer 9

-Genes can function within a given cell (autonomous) or influence surrounding cells (non-autonomous, e.g. secreted factor).
-Embryo transplantation studies to generate chimeras (organisms composed of a mix of cells from different sources)
- if there is a non-anonymous cell missing, can make more of that cell type

Question 10

Gene loss of function: Assessing cell death method

Answer 10

-Activated caspase 3 staining labels apoptotic cells, as it is a key converging factor that triggers DNA fragmentation
-Terminal dUTP nick-end labelling (TUNEL) marks DNA breaks (fragmentation)

Question 11

Cell cycle and proliferation identification method

Answer 11

-DNA replication factor cdt1 shows differential phase dependent expression to geminin
-Specific proteins are expressed at distinct phases and can be stained for immunhistochemically, e.g. phospho-histone 3 (mitosis), Ki-67 present in all cell phases except for G0 (quiescence)
-BrdU - thymidine analogue incorporates in newly synthesised DNA (regeneration)