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Biomedical technology > Recent advances in molecular medicine > Flashcards

Recent advances in molecular medicine Flashcards

1
Q

give an example of a disease caused by mutation or alteration in our DNA

A

cystic fibrosis
* A disease which affects 1 in 2500 people
* Caused by the inheritance of a defective CFTR
* CFTR -cystic fibrosis transmembrane Conductor regulator gene
* CFTR gene encodes for a CL ion transporter
* You can be a carrier if you only inherit one faulty gene

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2
Q

what is the causative gene in haemophilia?

A

factor VIII

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3
Q

what is the causative gen in inherited breast and ovarian cancer?

A

BRCA1/2

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4
Q

what is the causative gene in thalassemia?

A

alpha or beta globin

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5
Q

what is the causative gene in li-fraumeni syndrome?

A

p53

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6
Q

how was diabetes previously treated?

A

Previously treated with extracted insulin from the pancreas of cows and pigs. However this is slightly different than the human form of insulin, which can cause a whole load of side effects.

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7
Q

what is diabetes?

A

diabetes occurs when on emakes insufficient amouns of insulin

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8
Q

what is haemophillia

A

lack of or faulty factor VIII

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9
Q

how is haemophillia treated? what is wrong with this technique?

A

Haemophiliacs can be treated by giving purified factor VIII from volunteers
However it is difficult to purify and separate it from viral contaminants in the blood eg HIV, hepatitis
This is why many haemophiliacs have contracted AIDS or hepatitis from their supposedly pure preparation of human factor eight

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10
Q

what is a simple way of treating a disease that is caused by a specific protein deficit?

A

take a cell and isolate the causative gene, put it into an expression vector and then into the host cell where it will be intergrated into the genome so that the gene is expression

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11
Q

what is the easiest and cheapest cells to use to produce a specific protein?

A

bacterial cells

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12
Q

why are bacterial cells good host cells to use?

A

Easy and cheap to grow
Fast replication time (every 20 mins)

You can grow them in liquid media, in huge vessels
Really quick to get millions and millions of cells

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13
Q

what are the three most important signals in Ecoli for expressing a protein?

A
  1. The promoter
  2. The terminator
  3. The ribosome binding site
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14
Q

what is the promoter?

A

where RNA pol binds to start transcription

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15
Q

what is the promoter of a bacterial gene composed of?

A

Two highly conserved sequences at -35 and -10.

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16
Q

what forms the eukaryotic promoter?

A

one highly conserved sequence at -25 (the TATA box)

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17
Q

in order to get expression of a human gene in a bacterial cell, what do you need?

A

a bacterial promoter

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18
Q

what are the two considerations when choosign and using a promoter?

two types

A

a) Strong or weak promoter
b) Regulated promoter

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19
Q

why would someone opt for a strong or weak promoter?

A

Most people opt for a strong promoter to drive high levels of expression of the gene
Sometimes a weak one can be good because your product can be toxic to the bacterial cells, so you get slightly lower levels of expression so the product isnt so damaging

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20
Q

why would people opt for a regulated promoter?

A

a promoter than can switch on and off, can be useful when the product is toxic to the cells meaning that you have the option of growing up the bacterial cells to get millions and millions then switch on the promoter to express the gene in those cells. After 1-2 house lyse open and extract the protein, to optimise the yield.

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21
Q

give three examples of commonly used bacterial promoters

A
  1. Lac promoter
  2. Trp promoter
  3. Tac promoter
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22
Q

what is the Lac promoter?
how is it induced?

A

Controls transcription of the lacZ genes
It is inducible by IPTG

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23
Q

what is the Trp promoter?
how is it induced/ controlled?

A

Controls transcription of the trp operon
It is repressed by tryptophan, but induced by 3-indolylacetic acid

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24
Q

what is the Tac promoter?
how is it induced?

A

Man made, hybrid between trp and lac, combining their best features, is stronger than either of them.
The tac promoter/operator (dubbed PTAC) is one of the most widely used expression systems. Ptac is a strong hybrid promoter composed of the –35 region of the trp promoter and the –10 region of the lacUV5 promoter/operator.
Induced by IPTG, binds to to Lacl repressor

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25
Q

describe the terminator in bacteria

A

a sequence that can form a stem loop followed by a run of As in the template strand

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26
Q

what is the terminator?

A

The point at which transcription stops, found in the 3 prime control region of the gene (end).

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27
Q

describe the termination of transcription in a bacterial gene

A

In bacteria the terminator consists of a sequence that can form a stem loop followed by a run of As in the template strand
When transcribed into RNA is can form the stem loop structure - The non complementary bases form a loop in the middle.
As soon as this part of the DNA is transcribed to RNA, it immediately forms the stem loop causing the enzyme to pause
The enzyme pauses at the point where there is a run of As in the template strand and a run of Us in the newly synthesised RNA strand
AU base pairs are extremely unstable so when you have a whole load of them, the RNA DNA hybrid just falls apart.
A soon as this happens the enzyme will dissociate

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28
Q

what is the terminator in eukaryotic cells?

A

In eukaryotic cells there is no terminator, you get 3’ processing and polyadenylation
In the 3’ control region of human/animal gene, you see a set of conserved sequences (CPSF, poly A pol, CstF) which are bound by proteins, and cause the cleavage of the RNA after the CA and addition of a poly A tail.

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29
Q

what is the purpose of the poly A tail?

A

The poly A tail protects the RNA from degradation

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30
Q

what do you need if you want efficient termination of a human gene in a bacterial cell?

A

you need to supply a bacterial terminator

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31
Q

what is the ribosome binding site called in bacterial cells?

A

the Shine-Dalgarno sequence

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32
Q

how does the ribosome binding site in bacteria work?

A

the Shine-Dalgarno sequence is complementary to the end of the 16S ribosomal RNA part of the ribosome.
The ribosome binds to the ribosome binding site because of the 16S ribosomal RNA – then it initiates translation at the AUG usually 3-10 bases down stream

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33
Q

what is the ribosome binding site in eukaryotes?

A

Ribosomes attach to the 5’ end of messenger RNA and scans it looking for the first AUG initiation codon

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34
Q

if you want efficient translation of a human gene in a bacterial cell what do you need?

A

a bacterial ribosome binding site

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35
Q

why are specialised cloning vehicles made?

A

they allow you to place the foreign gene under control of a bacterial promoter, terminator and ribosome binding site.
To drive and optimise expression of your foreign gene of interest and facilitate production of the encoded protein

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36
Q

what should happen in the bacterial cell once the expression vector has been transfected?

A

the RNA pol in the bacterial cell should recognise the promoter at -35 and -10 and get transcription of the gene and termination at the terminator.
Ribosomes of the bacteria will bind to the ribosomal binding site and intiate at the AUG
(Engineer so that AUG is the first after the ribosomal binding site)
We should produce the protein that we want

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37
Q

why are fusion proteins used in expression vectors?

A

Sometimes our protein is degraded in bacterial cells, to prevent this degredation we create a fusion protein

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38
Q

what is a fusion protein?

A

A fusion protein is one which composes part of a bacterial protein at the N terminus (to stop the cell degrading it) and the protein that we want to express at the C terminus. This gene of interest is cloned in so that the reading frame is maintained.
This small amount of bacterial protein is enough to stop the degredation of our protein in these cells.

Along with the bacterial promoter and ribosome binding site.

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39
Q

what was the first fusion protein vector?

A

pGex vector

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40
Q

what did the pGex vector (plasmid DNA) contain?

A
  • ampicillin resistance gene
  • PPR322 an origin of replication
  • Bacterial promoter (tac)
  • Ribosome binding site
  • Coding region of the bacterial gene - glutathionine S transferase (GST)
  • MCS (multiple cloning sequence)
    o In this we have a sequence which encodes for the cleavage site for the factor 10 protease
    o Restriction sites into which we clone our gene
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41
Q

what is the purpose of an ampicillin resistance gene in the plasmid DNA vector?

A

allow selection of cells that have taken up the DNA

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42
Q

what is the origin of replication in bacteria?

A

ensures that every time bacteria divides it passes on a copy of the plasmid to the daughter cells

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43
Q

what is the purpose of multiple cloning sequence in plasmid DNA vector?

A

Where we find the restriction sequences into which we clone our gene

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44
Q

what are the advantages of using fusion vectors?

A
  • stability
  • ease of purification
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45
Q

How is the GST fusion protein purified?

A

GST binds with high affinity to glutathione
we can grow our bacterial cells in bulk, then lyse open the cells and pass the lysate through a column containing agarose beads linked to glutathione
the bacterial proteins should pass straight through but the fusion proteins will stick
add protease factor 10a which will cleave the (XA), its target site (in the fusion protein) - cleaving off the GST tag
releasing the human protein - almost purified

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46
Q

what is the recent, most commonly used fusion?

A

fusion with a metal chealate tag (poly histidine tag that binds to nickel columns)

protects from degredation and helps purify

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47
Q

what is an arabinose promoter?

what kind of promoter is it?

A

regulatory promoter
a bacterial promoter able to be switched on or off to drive expression of the gene when we want it too
By fusing the araBAD promoter to a gene of interest, the expression of the target gene can be solely regulated by arabinose

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48
Q

what is the purpose of an epitope in plasmid DNA vectors?

A

allows us to use a commercially available antibody to make sure that our protein is actually being expressed in these cells

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49
Q

what is the purpose of an enterokinase site in a plasmid DNA vector?

metal chealate

A

a protease site for the enterokinase enzyme that allows us to cleave away the polyHis tag from the protein of interest

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50
Q

what would the fusion protein of a metal chealate (poly his) plasmid look like?

A

Ending up with a fusion protein that has the polyHis tag (6xHis), an epitope, the cleavage (EK) site and mcs, followed by the protein we want

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51
Q

describe the process of purification using poly histidine tags

A

Grow in bulk, lyse cell, and pass the lysate through a column of nickel – all the bacterial protein pass through but the fusion protein will bind
Add histidine or imidazal which displaces the fusion protein from the column.
Our protein passes through
Then add enterokinase which will cleave the poly-his tag
Pass it back through a fresh column of nickel so that our protein will pass through and the poly-his tag will bind

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52
Q

what are four general problems assoviated with the production of recombinant proteins in E coli?

A
  • sometimes a human gene may contain a sequence that resembles a bacterial terminatory resulting in premature termination of transcription and truncated protein
  • codon usage: genetic code is universal however the frequency with which the codes are used depends upon the species
  • degredation of the protein by the bacteria
  • bacterial cells cannot glycosylate
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53
Q

how can we counter the general problem at the level of translation in the production of recombinant protein in e coli?

A

To solve this people will check the codons in the human gene – if there are infrequently used codons then they will mutate them to codons that are frequently used in bacterial cells
Eg mutate AGG to CGC – still code for the same amino acid

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54
Q

Frequency of the codons used is dependent upon…

A

…the abundance of the tRNAs in those cells

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55
Q

different codon bias in bacterial cells could result in what issue with the result recombinant protein?

A

Trying to express the gene (with infrequently used codons) in bacterial cells means we will get a much lower yield than we want and might even get truncated froms because the cell will runout of tRNA that recognise that codon.

the tRNA that recognise it are present in very low abundance

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56
Q

how do we counter the problem of degredation in recombinant proteins produced in e coli?

A

can use fusion vectors and ION- strains of bacteria (those that are protease deficient)

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57
Q

how do we solve the problem of processing in recombinant protein production in e coli?

A

bacterial cells cannot glycosylate, if glycosylation is necessary for protein function then you cannot make it in a bacterial cell
If a protein needs to be glycosylated, then you use higher eukaryotes – yeast and fungi can also be used as they glycosylate to a certain extent, but if there is a lot then animal cells are better

However with animal cells, they tend to want to grow attached to a matrix – (don’t grow in suspension making it harder to grow in bulk
They also divide fairly slowly – usually replicate every 18 hours

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58
Q

what are the most efficient hosts for human protein production?

A

higher eukaryotes

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59
Q

how are higher eukaryotic cells used in protein production?

A

multiple plates with a single layer of cells

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60
Q

what are the three kinds of promoters that can be used in higher eukaryotes?

A
  • viral promoters - eg CMV or SV40 (strongest promoters we know and work in virtually any animal cell)
  • heat shock promoter
  • mouse metallothionein promoter
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61
Q

what are heat shock promoters induced by?

A

induced at 40 degrees celcius

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62
Q

how are mouse metallothionein promoters induced?

A

induced by zinc

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63
Q

what are two issues with expression in animal cells?

A

relatively low and hard to grow in bulk

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64
Q

what are the components of the expression vector for mammalian cells?

A

(Cloning done in bacterial cell and then plugged into animal)
* ampicillin resistance gene
* bacterial origin of replication
* CMC promoter
* ATG start codon
* (followed by) signal sequence from immunoglobulin gene
* restriction sites
* Myc epitope
* 6x His
* Poly A tail
* mammalian selectable marker

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65
Q

what is the purpose of the signal sequence from immunoglobulin gene in the expression vector for mammalian cells?

A

to ensure that the protein is secreted out of the cell
so you can collect the protein from the media (no lysis or reseeding needed)

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66
Q

what is the purpose of the Myc epitope in the expression vector for mammalian cells?

A

to check for expression of the gene

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67
Q

what is the purpose of the 6x His tag in the expression vector for mammalian cells?

A

to allow us to purify our protein

68
Q

what are the advantages and disadvantages of mammalian cells used for protein production?

A
  • Best host for human proteins - expression generally good
  • Produce the same post translational modifications
  • Expression is generally low
  • Hard to grow in bulk
69
Q

what is Pharming?

A

Using animals themselves to produce a protein that we want
By altering an animal’s own DNA, or to splice in new DNA, called a transgene, from another species

In pharming, these genetically modified (transgenic) animals are used mostly to make human proteins that have medicinal value.

70
Q

where are transgene most commonly expressed in pharming?

A

o Eggs of chickens
o Milk of cows, goats or sheep
Take eggs and milk on daily basis and take protein from that

71
Q

when was the first transgenic farm animal?

A

in 1985
tracey a sheep expressing alpha 1 antitrypsin

72
Q

what are the four steps to produce a transgenic animal?

A

1) Preparation of transgene
2) Pronuclear microinjection
3) Transfer to pseudopregnant female
4) Screen offspring for presence of transgene

73
Q

give two examples of mammary gland specific promoters

A

casein or lactoglobulin – promoters only active in the mammary gland

74
Q

in the preparation of the transgene for pharming, after the gene is cloned in what happens?

A

cut away bulk of bacterial backbone to create a linear piece of DNA – effectively a transgene with the mammary gland promoter, signal sequence, gene, poly a site and selectable marker

75
Q

what are the advantages of having a linear piece of DNA?

A

more efficient at integrating into genome of host

76
Q

describe pronuclear microinjection

A

Take a newly fertilised egg, when egg and sperm have just fused but the nuclei are yet to.
Injection of DNA into male pronuclei
Injected DNA will integrate into the pronuclear DNA and upon fusion will be incorporated into the zygote
Culture the embryo, allow to grow and divide until it reaches the 16 cell morula stage
Implant into a pseudo pregnant female
Morula forms a blastocyst which implants into the uterus where it continues to grow into an embryo
End up with offspring

77
Q

what are the potential problems with pronuclear microinjection?

A
  1. Randomness of the insertion can have large effects on the level of expression – you only find out when the animal gets to maturity
  2. Can occasionally obtain a mosaic animal where the transgene is only present in a limited set of cells
    a. This occurs when integration is delayed until after the first cell division – transgene doesn’t integrate at this first stage but integrates at the 8 cell stage into only one of the cells so only an 8th of the cells in the mammary gland contain the transgene

Expression lower than you want and you don’t find out until they reach maturity

78
Q

what is the alternative to pronuclear microinjection that ensures high expression levels?

A

nuclear transfer

79
Q

even though the DNA in the cells is the same why are neurons and muscle cells structually and functionally distinct?

A

due to different DNA methylation patterns

80
Q

Low levels of methylation at the promoter means…

A

…RNA pol can bind and the gene is transcribed

81
Q

High levels of methylation at the promoter…

A

….block RNA pol and transcription factors from binding so the gene is switched off

82
Q

what is DNA methylation?

A
  • Addition of methyl (CH3) groups to DNA
  • Usually the C bases is methylated
83
Q

describe the changes in methylation patterns that occur in very early life

A

Upon fertilisation, the methylation marks on the DNA of the sperm are erased, followed by methylation marks on the oocyte being removed
Just before implantation there is a gradual increase in methylation – the genes that are not required inparticular cell types are methylated and turned off creating tissue specific patterns of gene methylation and expression
After this period of plasticity is over, it is thought that the methylation marks are relatively stable, locking these cells into their differentiated forms
Once established these methylation patterns are then stably maintained through the life of an organism

84
Q

describe why there are structural and functional differences between neurons and muscle cells?

A

In a neuron or muscle cell (unipotent cells) the level of methylation is much higher (than in pluripotent ones) and genes that are not required in these particular cells types are methylated and switched off
In a neuron genes involved in neuronal function will be unmethylated and active, and those involved in muscle contract are methylated and silent, the opposite is true in muscle cells

85
Q

what is the differnce between pluripotent and unipotent cells?

A

The only difference in the DNA is the level of methylation

86
Q

when was dolly the sheep cloned?

A

Dolly the sheep was successfully cloned in Britain in 1996 by the scientist “Ian Wilmut”.
Dolly was a genetic copy of the Finn Dorset ewe.
Her birth, more than 10 years ago showed that nuclei from specialized adult cells can be reprogrammed into all the cells of an organism.

87
Q

describe cloning by the Dolly-Roslin technique

A

isolate egg cells from ovaries and remove haploi nucleus using microinjection - scottish blackface ewe
isolate diploid somatic cels from mammary gland and induce quiescence (G0) by growth in low serum/nutrients
place together in culture and fuse cell membranes by electroporation
6 days in culture
uterine implantation
150 day gestation
birth

88
Q

breifly describe why nuclear transfer has higher expression than pronuclear microinjection

A
  • Grow mammary epithelial cells in culture
  • Introduce transgene into cells in culture – using the same construct
  • Select cell that has integrated transgene and those that are highly expressing the transgene !
  • Nuclei of only these cells which have taken up the transgene and integrated it are used
89
Q

what are theadvantages of the production of cloned animals which have originated from a single recombinant cell?

A

can create large number of animals each expressing identical levels of a therapeutic protein - we can guarantee that offspring are expressing high level of your protein because you selected these cells specifically
no reseeding necessary

90
Q

what expression system should be used if the protein of interest is unglycosylated?

A

easiest and cheapest is bacterial cells

91
Q

what expression system should be used if the protein is glycosylated?

A

animal cells or animals themselves

92
Q

how is insulin made in the body?

A

made as preproinsulin which is then folded and cleaved
the alpha and beta chains are held together by disulphide bridges

93
Q

is insulin glycosylated or not?

A

insulin is not glycosylated

93
Q

is insulin glycosylated or not?

A

insulin is not glycosylated

94
Q

how has insulin be previously obtained and why were new methods needed?

A
  • insulin was obtained from the pancreas of cows and pigs
  • the side effects

The most common adverse reactions reported with this insulin include hypoglycemia, allergic reactions, injection site reactions, lipodystrophy, weight gain, and edema

95
Q

describe the steps to producing synthetic insulin

A

Chemical synthesise a synthetic gene encoding the A chain and a synthetic gene encoding the B chain
Then fuse these synthetic gene to the coding region of lacZ, which encodes beta gal to ensure that it is stable in the cells

Put these constructs into bacterial cells and get expression – beta gal followed by the A chain and beta gal followed by the B chain
Add cyanogen bromide to cut the internal methionines – the only internal methionine is at the start of the A chain and the B chain – allows the beta gal tag to be cleaved away
Purify the A and B chain and added the disulphide bridges

The final steps are to collect the bacteria, break open the cells, and purify the insulin protein expressed from the recombinant human insulin gene.
Humulin

96
Q

what is factor VIII?

A

The F8 gene provides instructions for making a protein called coagulation factor VIII playing a central role in blood clotting

97
Q

how is factor VIII previously produced?

A

purification from human blood

98
Q

what is the problem with getting factor VIII from purified human blood?

A
99
Q

give 3 features of factor VIII that make it difficult to make

A
  • very large over 186kb
  • contains 17 disulphide bridges
  • only active after extensive glycosylation
100
Q

is factor VIII glycosylated or not?

A

factor VIII is extensively glycosylated

(only active after it is)

101
Q

cell culture costs ____ per gram of protien

A

$100

102
Q

pharming can cost ____ per gram

A

$2 - $20

103
Q

what are the most efficient bioreactors?

A

cows
8000l of milk per yr with 40 to 80kg of protein

104
Q

what is the most common hemophilia deficiency?

A

factor VIII

105
Q

what animals are used to produce alpha 1 antitrypsin for treatment of emphysema?

A

sheep

106
Q

what animals are used to produce CFTR for treatment of cystic fibrosis?

A

sheep

107
Q

what animals are used to produce tissue plasminogen activator for treatment of thrombosis?

A

sheep
pig

108
Q

what animals are used to produce factor VIII for treatment of hemophilia?

A

sheep
pig
cow

sheep and pig also used to make factor XI

109
Q

what animals are used to produce fibrinogen to treat wound healing?

A

sheep
cow

110
Q

what animals are used to produce human protein c for treatment of thrombosis?

A

goat

111
Q

what animals are used to produce antithombin 3 for treatment of thrombosis?

A

goat

112
Q

what animals are used to produce glutamic acid decarboxylase for treatment of type 1 diabetes?

A

goat

113
Q

what animals are used to produce Pro542 for treatment of HIV?

A

goat

114
Q

what animals are used to produce alpha lactalbumin for anti infection?

A

cow

115
Q

what animals are used to produce collagen 1 and 2 for tissue repair/ treatment of arthritis?

A

cow

116
Q

what animals are used to produce collagen 1 and 2 for tissue repair/ treatment of arthritis?

A

cow

117
Q

what is biosteel?

A

BioSteel™ Goats have been genetically modified to produce the protein from Golden Orb Weaver Spider (Nephila clavipes) silk in their milk

 spider silk is stronger and more flexible then steel and offers a lightweight alternative to carbon fibre

118
Q

what conditions could stem cells possibly treat?

A
  • Spinal cord injuries
  • Parkinsons disease,
  • AD
  • Type 1 diabetes
  • CVD
  • deafness
119
Q

how do stem cells avoid senescence?

A

they express high levels of telomerase

120
Q

describe the terms unipotent, multipotent, pluripotent and totipotent

A
  • Unipotent stem cells form only one type of specialised cell
  • Muiltpotent stem cells can form multiple types of cells and tissues
  • Pluripotent stem cells can form most or all cell types in the adult
  • Totipotent stem cells can form all adult cell types as well as the specialised tisses to support the development of the embryo
121
Q

when do cells in the embyro transition from totipotent to pluripotent?

A

From the zygote to the morula we would class the cells as totipotent
From the blastocyst, the inner cell mass cells are pluripotent

122
Q

where do embryonic stem cells come from?

A

the inner cell mass from blastocysts left over from invitro fertilisation in the lab or aborted fetuses

123
Q

what is the key regulator of potency?

A

Oct 4

124
Q

where have adult stem cells been found?

A

o Stem cells have been found in the blood, bone marrow, liver, kidney, cornea, dental pulp, umbilical cord, brain, skin, muscle salivary gland and many other places

125
Q

give 4 features of Oct 4

domain, binding location, expression, associate proteins

A
  • POU domain transcription factor
  • Binds to DNA through the AGTCAAAT consensus motif
  • Expressed exclusively in stem cells
  • Works in conjunction with nanog and sox2 to maintain toti/pluripotency
126
Q

where do we find high levels of Oct 4 in the fertilised egg/embryo

A

we find high levels of Oct 4 in the zygote, morula and inner cell mass

when oct 4 levels decrease they allow the TE to form.

127
Q

what happens to oct4 when cells begin to differentiate?

A

Oct4 will go down

128
Q

what two jobs is Oct4 doing in stem cells?

A

activate self renewal genes and repress/silence developmental control genes

129
Q

what are the two sources of embryonic stem cells?

A
  1. Excess fertilised eggs from IVF clinics
  2. Therapeutic cloning
130
Q

what promoters of self renewal genes do Oct4, Nanog and Sox2 activate?

A

Oct4 Sox2 Nanog C-Myc Myst3

131
Q

what promoters of developmental genes do Oct 4 Nanog and Sox2 silence?

A

Neurog1, Pax6, Gata4, Cdx2

132
Q

can therapeutic cloning be used to produce stem cells?
what benefits and ethical considerations do they have?

A

Yes it can be used - adult differentiated cell and enucleated oocyte fused to produce cloned zygote, culture and matured to blastocyst and harvest stem cells from ICM
To provide stem cells that are a genetic match to the patient
Ethically - we are creating embryos/life just to use as spare parts, potential for human cloning aswell

133
Q

what is the primary role of adult stem cells?

A

present in very small numbers their primary role is to maintain and repair tissues in which they are found
they remain quiescent for long periods until activated, when they differentiate into the cell type consistant with the signals ssent by the environment

134
Q

where are adult stem cell primarily isolated from?

A

bone marrow
(they are easier to activate to some cell types than others)

135
Q

what do heamatopoietic stem cells differentiate into ?

A

all types of blood cells

136
Q

what do mesenchymal stem cells differentiate into?

A

bone, cartilage and fat

137
Q

what do umbilical cord stem cells differentiate into ?

A

blood bone and neurons

138
Q

what do neural stem cells differentiate into ?

A

neurons, astrocytes and oligodendrocytes

139
Q

compare embryonic stem cells with adult stem cells

3

A

embryonic stem cells are pluripotent (can become any cell), they are stable (can undergo many cell divisions), easy to obtain but blastocyst is destroyed

Adult stem cells are multipotent (can become many but not any), they are less stable (capacity for self renewal is limited, difficult to isolate in adult tissue

140
Q

when were induce plurpotent stem cells first developed?

A

in 2007

141
Q

application of what four pluripotency factors are used to convert an adult cell back into an embryonic stem cell?

A

Oct4, Sox2, c-Myc , KLF4

142
Q

what other combinations of plurpotency factors work to produced iPS cells?

A

o Oct4, Sox2, c-Myc , KLF4
- IPS efficiency highest when all 4 TF used together, although cmyc and klf4 are dispensable. – and oct4??
- Oct4, sox2, nanog, lin28 this combination also works
- When all six are used – it enhanced the formation

143
Q

what combination of plurpotency factors enhances the formation of iPS cells?

A

Oct4, nanog, sox2, lin28, cmyc and klf4

144
Q

how much does combination of all 6 pluripotency factors enhance iPS fromation?

A

enhances iPS formation 10 fold and reduces time of reprogramming from 26 days to 17 days

145
Q

what can influence the differentiation capacity of resultant iPS cells?

A

the parental cell

there is a memory of the original cell type
Application of demethylating agents to iPS cells to completely wipe out the methylation marks to get fully pluripotent stem cells

Eg. mouse bone-marrow-derived and B-cell-derived iPS cells showed more efficient differentiation along haematopoietic lineages than fibroblast or neural iPS cell lines.

146
Q

how are iPS cells created?

A

isolate cells from patietn (skin or fibroblasts) grow in a dish
treat cells with reprogramming factors
wait a few weeks
pluripotent stem cells

change culture condition to stimulate cells to differentiate into a variety of cell types

147
Q

how can iPS cell production be used to correct genetic defects/ diseases caused by mutation?

A

take cell from patient, revert to pluripotency, correct the mutation (CRISPR-cas9), grow and differentiate the cell we need and put back into patient – allows us to correct a genetic defect

148
Q

what are the potential uses of iPS cells?

A

drug modelling, disease modelling, quality control for safety and efficacy (cell identity, purity, and potency) and cell based therapy

149
Q

what are the pros of iPS cell technology?

A

Pros:
– Cells would be genetically identical to patient or donor of skin cells (no immune rejection!)
– Do not need to use an embryo

150
Q

what are the cons to iPS cell technology?

A

cons
– Cells would still have genetic defects – unless corrected by gene editing
– One of the pluripotency genes c-myc is a cancer gene (oncogene) – differentiate first in vitro and only put the fully differentiated ones back into the patient
– Depending of cell of origin degree of differentiation varies
– Low survival rates

151
Q

describe the stem cell trials for cardiac disease

A

Induce heart attack in female mouse
Take mesenchymal stem cells from male mouse (so can follow in the female)
Injected into damaged area
Mesenchymal stem cell differentiated into cardiac muscle cells, they formed electrical connections with healthy muscle cells and improved heart function by 35%

152
Q

what was the outcome of the stem cell trials for cardiac disease in humans?

A
  • number of trials underway involving mesenchymal stem cells
  • one randomised double blind trial showed ongoing benefit up to 18 months
    – better mean reduction in infarct size
    – better improvement in LV perfusion
    – no major adverse events
  • larger, randomised trial now underway
153
Q

how can be stem cells be used to treat diabetes?

A

creating functional beta cells and putting them into pancreas
apply soluble inductive signals (small molecules, proteins) to human ES or iPS cells to differentiate them into stem cell beta cell

human ES or iPS cells -> definitive endoderm -> pancreatic progenitor -> endocrine progenitor -> stem cell beta cell

154
Q

what is the molecular basis of diabetes?

A

reduced pancreatic beta cell mass causing insufficient production of insulin resulting in uncontrolled glucose levels
reduced beta cell mass provoked by: impaired beta cell regeneration or aggrevated beta cell death

type1: autoimmune destruction of beta cells
type 2: progressive beta cell failure

155
Q

how can we enhance the ability of stem cells to get to their required site of action?

A

pretreatment in the implantation sites to induce angiogenesis to create vascular networks and develop less intolerant environment for implantaion of the cells

putting the stem cells within a semi permeable membrane device and then subcutaneous or kidney capsule implantation

improves long term surivival, differentiation maturation and glucose responsiveness

156
Q

what two ways can iPS cells be created/implanted for diabetes?

A

iPS cells in monolayer culture or suspension, then application of factors to creat endocrine progenitors which are transplanted, in vivo differentiation and maturation, islet like cells then insulin secretion and reversal of hyperglycemia

iPS cells in low adhesion culture or suspension, application of factors to creat endocrine progenitors, in vitro differentiation and maturation to create pancreatic beta cells with matural beta cell marks and glucose responsive, then transplantation and further maturation in vivo. resulting in matural beta cell markers, insulin secretion, ameliorates hyperglycemia (in diabetic mice)

157
Q

how does IPTG induce expression by the Lac promoter (operon)?

A

IPTG (Isopropyl ß-D-1-thiogalactopyranoside), is a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon and it is therefore used to induce protein expression where the gene is under the control of the lac operator

158
Q

how much more efficient is the tac promoter?

A

The tacI and the tacII promoters respectively direct transcription approximately 11 and 7 times more efficiently than the derepressed parental lac UV5 promoter
and
approximately 3 and 2 times more efficiently than the trp promoter in the absence of the trp repressor.

159
Q

what represses the Tac promoter?
how is expression controlled?

A

The expression of PTAC is repressed by the lacI protein. The lacIq allele is a promoter mutation that increases the intracellular concentration of LacI repressor, resulting in the strong repression of PTAC.
The addition of the inducer IPTG inactivates the LacI repressor. Thus, the amount of expression from PTAC is proportional to the concentration of IPTG added: low concentrations of IPTG result in relatively low expression from PTAC and high concentrations of IPTG result in high expression from PTAC.

160
Q

what is polyadenylation?

A

Polyadenylation is the post-transcriptional additional of multiple adenine (A) nucleotides to the tail of a messenger RNA transcript. The purpose and mechanism of polyadenylation vary across cell types, but polyadenylation generally serves to promote transcript longevity in eukaryotes and promote transcript degradation in prokaryotes.

in eukaryotes, The addition of the poly(A) tail is important for stability of the mRNA, protection from degradation, and is integral to the nuclear export and translation processes as well

161
Q

what is factor Xa?

A

The coagulation factor Xa is a serine protease which. recognizes the amino acid sequence. — Ile — Glu — Gly — Arg — with a high degree of specificity.
The cleavage of this sequence activates the natural substrate prothrombin to thrombin

restriction site in pGex vector

162
Q

how is pGex vector protein purified?

A

GST helps us purify the protein
o Because GST binds with high affinity to glutathione
 So we can grow our bacterial cells in bulk, then lyse open the cells and pass the lysate through a column (containing agarose beads linked to glutathione)
* So the bacterial proteins should pass straight through but the fusion proteins will stick
o Add preotease factor 10a which will cleave the (XA) its target site (in the fusion protein) - cleaving of the GST tag
 Releasing the human protein – almost purified the protein

163
Q

what was the first vector of the type that helped increase the stability of the protein while helping purify it as well?

A

pGex vector

164
Q

what is required for the recombinant protein to be secreted into milk?

A

Secretion of the recombinant protein into the milk requires an amino-terminal signal peptide, which directs the nascent polypeptide into the endoplasmic reticulum. Via the Golgi-apparatus, the matured proteins are transported into secretory vesicles, which fuse with the cell membrane and release their cargo into the lumen of the mammary gland.

165
Q

whats needed for recombinant protein expression in the mammary gland? give three examples

A

Mammary gland specific promoter and regulatory elements, such as casein, lactoglobulin and lactoalbumin promoter elements were used to target the expression of a recombinant protein to the mammary epithelium during the lactation period.

166
Q

why is the nuclear DNA injected into the male pornucleus in pronuclear microinjection?

A

It is generally accepted that microinjection into the male pronucleus (contributed from the sperm) results in a higher rate of success in terms of transgenic offspring produced. The male pronucleus is generally the larger of the two pronuclei present and is typically located at the pe

Biomedical technology (9 decks)

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