L2 + 3 Principles and Techniques Flashcards
Define pattern formation
The process by which cells are organised in space and time to produce a well-ordered strucuture within the embryo
What are the 3 axis of the body
What is X Y and Z
A-P
D-V
L-R
AP (X) DV (Y) LR (Z)
What is the head tail axis
Which is head
Which is tail
AP
Head = anterior
Tail = Posterior
What axis is front to back
Which is back
Which is front
DV
Dorsal is back (dorsum)
Ventral is front
Define morphogenesis
Cell and tissue movement and changes in cell behaviour which give the developing embryo its shape in 3 dimensions
What are the four things which contribute to morphogenesis
Changes in;
Adhesion, shape, death and migration
What occurs to cells in order for the digits to form properly
Cell death
Describe differentiation
Process by which cells become different from each other and acquire specilaised properites. This is governed by changes in gene expression which dictate the repertoire of genes expressed
Over time what happens to a cells specilisation and potency
Potency becomes restricted as cell becomes more differentiated
The gradual process of differentiation involves what steps
Egg/Stem Specification Determination Differentiation Maturation
Which word describes irreversible comittment of a cell to its fate
Determination
How can you test if a cell is determined
Transplant - see if it differentiates based on new or old position - if old then cell is determined
How do muscle cells show and example of maturation
They express contractile proteins but it is only after innervation that the type of fibre is determined
Define cell growth
Increase in mass or size
Was does growth rate vary according to
Age and type of organ
What are the three methods of cellular growth
Cell proliferation
Cell enlargement
Growth by accretion
What is growth by accretion
Association with ECM proteins increasing the distance between two cells and subseuently causing growth
Who proposed the funnel model
Haeckel
Describe the funnel model
Initatially all organism are similar and then over time the organisms gradually become more different
Who proposed the hourglass model
Von Baer
Describe the hourglass model
Early events - such as gastrulation- are different
Intermediate stages are similar
Then become different at later stages
Which is accepted … hourglass or funnel
Hourglass
Since early events such as gastulation are different
Which techniques may be used to investigate where and when a gene is expressed
In-situ hybridisation Northern blot RT-PCR Microarray Reporter transgenic lines
Northern blots and RT-PCR may be used to analyse where and when a gene is expressed upon what conditions
If only a specific tissue is used to provide the mRNA
If the whole cell was used then it would give no information as to the location of the gene in the embryo
What techniques would provide information of the spatial expression of a gene
In situ
Northern blot (condit)
RT-PCR (condit)
Reporter lines
What must be known in order to produce a trangenic reporter line
How transcription of the gene is controlled - enhancer and promoter regions
Describe how a transgenic reporter line produced
Expression of a gene determined by regulatory regions
Gene is replaced by a reporter gene
Reporter gene will be expressed wherever the gene is usually expressed
What is the differnce between fusion with GFP
With fusion a GFP tag is fused in frame which allows visualisation of the PROTEIN compared to the transgenic reporter line allows the location of the mRNA to be visualised
Describe the process of a microarray analysis
cDNA for every gene into a spot on the grid
mRNA is isolated and labelled
will hybridise if gene present
This can then be detected
What does an in-situ hybridisation reveal
Location of mRNA
What is the probe in an in-situ hybridisation labelled with
DIG
What binds to the DIG labelled probe
Anti-DIG
What two reporter genes may be used
GFP and beta-galactasidease
What colourr signal does alkaline phosphaase give
Blue
Where is the colour in an in-situ hybridisation seen
Wherever the anti-DIG hybridies to DIG labelled probe which has hybridised to the mRNA
What is a microarray analysis an example of
A genome wide approach
In a microarray what is fixed to the grid - what then hybridises
cDNA antisense to the mRNA
mRNA would hybridise if the gene was expressed in that tissue type
How can an microarray be used to compare transcriptomes of two tissues
Label the mRNA from cell one with colour 1
Label mRNA from cell two with colour 2
Where colour one shown ==> gene only expressed in cell one
Where colour two shown ==> gene only expressed in cell two
Blank = no gene
Hybrid of colours ==> Gene expressed in both of the cells
What is the main requirement for microanalysis
Need a large ammount of mRNA
So young/small embryos would be unsuitable
What is the technique developed from microarray
RNAseq
Instead of hybdridation sequence at the end of fragment to determine the gene
What is an advantage of RNAseq compared with microarrary analusus
Can do RNAseq with much less mRNA - future
Immunodetection methods include
Immunohistochemistry and immunoflourexecnece
What are the pre-requisites for immuno-detection
Prior knowledge of protein
AND
A specific AB availble which would recognise the protein
Describe the method for immunodetection
Take section of tissue and incubate in presence of antibody
Use seconddary antibody which is conjugated so can be detected
What is the secondary antobdy
anti-IgE
What is the purpose of the seconadry antibody
Amplification of the signal
How many GFP be used to visualise where the protein is expressed in a cell
Fuse GFP in frame with the gene of interest
But the gene of interest is left intact
THIS IS DIFFERENT FROM A TRANSGENIC REPORTER LINE
Define loss of function
A mutation in a gene that disrupts the expression or the fuction of the protein product encoded by the gene
What are the techniques to investigate loss of function
Forward genetics and reverse genetics
What is forward genetics
What organisms are used
Seeks to identify a gene whose mutation caused a particular phenotype
Drosoph, zebrafish and mice
What is reverse genetics
Seeks to characterise the phenotype
Describe the typical steps of a mutagenesis screen
Randomly mutagenise the males
Cross mutagenised males with wt females
Cross again with wt femals
Incross this family
Describe the method for creating a conventional knockout organism
Inject construct using HR to disrupt the gene
Inject into mES cells
Select transformed cells
Inject transformed cells into inner cell mass of the blastocyst
Implant into mouse
Formaiton of chimera
Breed for germline transmission
Result is a mouse in which every cell contains the disrupted gene
Describe the method for generating a tissue specific conditional knockout
Gene of interst is floxed
SAME STEPS TO GET THE MOUSE WHERE ALL OF THE CELLS CONTAIN THE FLOXED VERSION OF THE GENE
Another mouse should have cre expressed under a certain promoter
Cross
Where conditions of cre promoter met - cre expressed
Cre cleaves inbetween loxP sites and gene is lost
When is condition knockouts important
When the gene of interest is essential for early development
E.g. a conditional knockout would kill the cell
Descirbe the experiement where the ZPA was grafted into an ectopic location
Resulted in duplication of the digits with symmetry
Describe the spemann mangold organiser graft
Graft organiser into an ectopic location (on the ventral side)
Leads to duplication of the axis
Concluded that the region has an organiser capacity
What is significant which means that quail and chick can be used for studies
They are very similar and antibodies can be used that recognise proteins at the surface of the quail but not the chick
Desribe the chick/quail studies
Take tissues from quail and transpant into the chick
Use immunohistochemistry to visualuse the quail cells - see what they have become
Why are chick/quail studies restrictive?
Cant get single cell resolution
Will always be around 50-100
Describe the brainbow technique
Use of homologous recombination to insert various fluorescent genes each of which are flanked by loxP sites - each loxP site is slightly different so is recognised by a different cre recombinase
Can only have one type of excision - this will depend on the cell type and leave a certain set of colors left once crossed with cre recombinase
