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BMS242 Developmental Biology > L2 + 3 Principles and Techniques > Flashcards

L2 + 3 Principles and Techniques Flashcards

1
Q

Define pattern formation

A

The process by which cells are organised in space and time to produce a well-ordered strucuture within the embryo

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2
Q

What are the 3 axis of the body

What is X Y and Z

A

A-P
D-V
L-R

AP (X) DV (Y) LR (Z)

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3
Q

What is the head tail axis

Which is head

Which is tail

A

AP

Head = anterior

Tail = Posterior

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4
Q

What axis is front to back

Which is back

Which is front

A

DV

Dorsal is back (dorsum)

Ventral is front

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5
Q

Define morphogenesis

A

Cell and tissue movement and changes in cell behaviour which give the developing embryo its shape in 3 dimensions

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6
Q

What are the four things which contribute to morphogenesis

A

Changes in;

Adhesion, shape, death and migration

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7
Q

What occurs to cells in order for the digits to form properly

A

Cell death

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8
Q

Describe differentiation

A

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

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9
Q

Over time what happens to a cells specilisation and potency

A

Potency becomes restricted as cell becomes more differentiated

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10
Q

The gradual process of differentiation involves what steps

A 
Egg/Stem 
Specification
Determination
Differentiation
Maturation
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11
Q

Which word describes irreversible comittment of a cell to its fate

A

Determination

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12
Q

How can you test if a cell is determined

A

Transplant - see if it differentiates based on new or old position - if old then cell is determined

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13
Q

How do muscle cells show and example of maturation

A

They express contractile proteins but it is only after innervation that the type of fibre is determined

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14
Q

Define cell growth

A

Increase in mass or size

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15
Q

Was does growth rate vary according to

A

Age and type of organ

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16
Q

What are the three methods of cellular growth

A

Cell proliferation
Cell enlargement
Growth by accretion

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17
Q

What is growth by accretion

A

Association with ECM proteins increasing the distance between two cells and subseuently causing growth

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18
Q

Who proposed the funnel model

A

Haeckel

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19
Q

Describe the funnel model

A

Initatially all organism are similar and then over time the organisms gradually become more different

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20
Q

Who proposed the hourglass model

A

Von Baer

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21
Q

Describe the hourglass model

A

Early events - such as gastrulation- are different
Intermediate stages are similar
Then become different at later stages

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22
Q

Which is accepted … hourglass or funnel

A

Hourglass

Since early events such as gastulation are different

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23
Q

Which techniques may be used to investigate where and when a gene is expressed

A 
In-situ hybridisation 
Northern blot 
RT-PCR
Microarray 
Reporter transgenic lines
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24
Q

Northern blots and RT-PCR may be used to analyse where and when a gene is expressed upon what conditions

A

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

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25
What techniques would provide information of the spatial expression of a gene
In situ Northern blot (condit) RT-PCR (condit) Reporter lines
26
What must be known in order to produce a trangenic reporter line
How transcription of the gene is controlled - enhancer and promoter regions
27
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
28
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
29
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
30
What does an in-situ hybridisation reveal
Location of mRNA
31
What is the probe in an in-situ hybridisation labelled with
DIG
32
What binds to the DIG labelled probe
Anti-DIG
33
What two reporter genes may be used
GFP and beta-galactasidease
34
What colourr signal does alkaline phosphaase give
Blue
35
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
36
What is a microarray analysis an example of
A genome wide approach
37
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
38
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
39
What is the main requirement for microanalysis
Need a large ammount of mRNA So young/small embryos would be unsuitable
40
What is the technique developed from microarray
RNAseq | Instead of hybdridation sequence at the end of fragment to determine the gene
41
What is an advantage of RNAseq compared with microarrary analusus
Can do RNAseq with much less mRNA - future
42
Immunodetection methods include
Immunohistochemistry and immunoflourexecnece
43
What are the pre-requisites for immuno-detection
Prior knowledge of protein AND A specific AB availble which would recognise the protein
44
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
45
What is the secondary antobdy
anti-IgE
46
What is the purpose of the seconadry antibody
Amplification of the signal
47
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
48
Define loss of function
A mutation in a gene that disrupts the expression or the fuction of the protein product encoded by the gene
49
What are the techniques to investigate loss of function
Forward genetics and reverse genetics
50
What is forward genetics What organisms are used
Seeks to identify a gene whose mutation caused a particular phenotype Drosoph, zebrafish and mice
51
What is reverse genetics
Seeks to characterise the phenotype
52
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
53
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
54
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
55
When is condition knockouts important
When the gene of interest is essential for early development E.g. a conditional knockout would kill the cell
56
Descirbe the experiement where the ZPA was grafted into an ectopic location
Resulted in duplication of the digits with symmetry
57
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
58
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
59
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
60
Why are chick/quail studies restrictive?
Cant get single cell resolution | Will always be around 50-100
61
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

BMS242 Developmental Biology (17 decks)

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