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

L2 + 3 Principles and Techniques Flashcards

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AP® Biology flashcards Biology 101 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
Q

What techniques would provide information of the spatial expression of a gene

A

In situ
Northern blot (condit)
RT-PCR (condit)
Reporter lines

26
Q

What must be known in order to produce a trangenic reporter line

A

How transcription of the gene is controlled - enhancer and promoter regions

27
Q

Describe how a transgenic reporter line produced

A

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
Q

What is the differnce between fusion with GFP

A

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
Q

Describe the process of a microarray analysis

A

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
Q

What does an in-situ hybridisation reveal

A

Location of mRNA

31
Q

What is the probe in an in-situ hybridisation labelled with

A

DIG

32
Q

What binds to the DIG labelled probe

A

Anti-DIG

33
Q

What two reporter genes may be used

A

GFP and beta-galactasidease

34
Q

What colourr signal does alkaline phosphaase give

A

Blue

35
Q

Where is the colour in an in-situ hybridisation seen

A

Wherever the anti-DIG hybridies to DIG labelled probe which has hybridised to the mRNA

36
Q

What is a microarray analysis an example of

A

A genome wide approach

37
Q

In a microarray what is fixed to the grid - what then hybridises

A

cDNA antisense to the mRNA

mRNA would hybridise if the gene was expressed in that tissue type

38
Q

How can an microarray be used to compare transcriptomes of two tissues

A

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
Q

What is the main requirement for microanalysis

A

Need a large ammount of mRNA

So young/small embryos would be unsuitable

40
Q

What is the technique developed from microarray

A

RNAseq

Instead of hybdridation sequence at the end of fragment to determine the gene

41
Q

What is an advantage of RNAseq compared with microarrary analusus

A

Can do RNAseq with much less mRNA - future

42
Q

Immunodetection methods include

A

Immunohistochemistry and immunoflourexecnece

43
Q

What are the pre-requisites for immuno-detection

A

Prior knowledge of protein
AND
A specific AB availble which would recognise the protein

44
Q

Describe the method for immunodetection

A

Take section of tissue and incubate in presence of antibody

Use seconddary antibody which is conjugated so can be detected

45
Q

What is the secondary antobdy

A

anti-IgE

46
Q

What is the purpose of the seconadry antibody

A

Amplification of the signal

47
Q

How many GFP be used to visualise where the protein is expressed in a cell

A

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
Q

Define loss of function

A

A mutation in a gene that disrupts the expression or the fuction of the protein product encoded by the gene

49
Q

What are the techniques to investigate loss of function

A

Forward genetics and reverse genetics

50
Q

What is forward genetics

What organisms are used

A

Seeks to identify a gene whose mutation caused a particular phenotype

Drosoph, zebrafish and mice

51
Q

What is reverse genetics

A

Seeks to characterise the phenotype

52
Q

Describe the typical steps of a mutagenesis screen

A

Randomly mutagenise the males
Cross mutagenised males with wt females
Cross again with wt femals
Incross this family

53
Q

Describe the method for creating a conventional knockout organism

A

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
Q

Describe the method for generating a tissue specific conditional knockout

A

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
Q

When is condition knockouts important

A

When the gene of interest is essential for early development

E.g. a conditional knockout would kill the cell

56
Q

Descirbe the experiement where the ZPA was grafted into an ectopic location

A

Resulted in duplication of the digits with symmetry

57
Q

Describe the spemann mangold organiser graft

A

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
Q

What is significant which means that quail and chick can be used for studies

A

They are very similar and antibodies can be used that recognise proteins at the surface of the quail but not the chick

59
Q

Desribe the chick/quail studies

A

Take tissues from quail and transpant into the chick

Use immunohistochemistry to visualuse the quail cells - see what they have become

60
Q

Why are chick/quail studies restrictive?

A

Cant get single cell resolution

Will always be around 50-100

61
Q

Describe the brainbow technique

A

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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