Recent advances in molecular medicine Flashcards
(167 cards)
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
2
Q
what is the causative gene in haemophilia?
A
factor VIII
3
Q
what is the causative gen in inherited breast and ovarian cancer?
A
BRCA1/2
4
Q
what is the causative gene in thalassemia?
A
alpha or beta globin
5
Q
what is the causative gene in li-fraumeni syndrome?
A
p53
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.
7
Q
what is diabetes?
A
diabetes occurs when on emakes insufficient amouns of insulin
8
Q
what is haemophillia
A
lack of or faulty factor VIII
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
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
11
Q
what is the easiest and cheapest cells to use to produce a specific protein?
A
bacterial cells
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
13
Q
what are the three most important signals in Ecoli for expressing a protein?
A
- The promoter
- The terminator
- The ribosome binding site
14
Q
what is the promoter?
A
where RNA pol binds to start transcription
15
Q
what is the promoter of a bacterial gene composed of?
A
Two highly conserved sequences at -35 and -10.
16
Q
what forms the eukaryotic promoter?
A
one highly conserved sequence at -25 (the TATA box)
17
Q
in order to get expression of a human gene in a bacterial cell, what do you need?
A
a bacterial promoter
18
Q
what are the two considerations when choosign and using a promoter?
two types
A
a) Strong or weak promoter
b) Regulated promoter
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
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.
21
Q
give three examples of commonly used bacterial promoters
A
- Lac promoter
- Trp promoter
- Tac promoter
22
Q
what is the Lac promoter?
how is it induced?
A
Controls transcription of the lacZ genes
It is inducible by IPTG
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
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
25
describe the terminator in bacteria
a sequence that can form a stem loop followed by a run of As in the template strand
26
what is the terminator?
The point at which transcription stops, found in the 3 prime control region of the gene (end).
27
describe the termination of transcription in a bacterial gene
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
28
what is the terminator in eukaryotic cells?
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.
29
what is the purpose of the poly A tail?
The poly A tail protects the RNA from degradation
30
what do you need if you want efficient termination of a human gene in a bacterial cell?
you need to supply a bacterial terminator
31
what is the ribosome binding site called in bacterial cells?
the Shine-Dalgarno sequence
32
how does the ribosome binding site in bacteria work?
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
33
what is the ribosome binding site in eukaryotes?
Ribosomes attach to the 5’ end of messenger RNA and scans it looking for the first AUG initiation codon
34
if you want efficient translation of a human gene in a bacterial cell what do you need?
a bacterial ribosome binding site
35
why are specialised cloning vehicles made?
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
36
what should happen in the bacterial cell once the expression vector has been transfected?
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
37
why are fusion proteins used in expression vectors?
Sometimes our protein is degraded in bacterial cells, to prevent this degredation we create a fusion protein
38
what is a fusion protein?
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.
39
what was the first fusion protein vector?
pGex vector
40
what did the pGex vector (plasmid DNA) contain?
- 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
41
what is the purpose of an ampicillin resistance gene in the plasmid DNA vector?
allow selection of cells that have taken up the DNA
42
what is the origin of replication in bacteria?
ensures that every time bacteria divides it passes on a copy of the plasmid to the daughter cells
43
what is the purpose of multiple cloning sequence in plasmid DNA vector?
Where we find the restriction sequences into which we clone our gene
44
what are the advantages of using fusion vectors?
* stability
* ease of purification
45
How is the GST fusion protein purified?
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
46
what is the recent, most commonly used fusion?
fusion with a metal chealate tag (poly histidine tag that binds to nickel columns)
| protects from degredation and helps purify
47
what is an arabinose promoter?
| what kind of promoter is it?
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
48
what is the purpose of an epitope in plasmid DNA vectors?
allows us to use a commercially available antibody to make sure that our protein is actually being expressed in these cells
49
what is the purpose of an enterokinase site in a plasmid DNA vector?
| metal chealate
a protease site for the enterokinase enzyme that allows us to cleave away the polyHis tag from the protein of interest
50
what would the fusion protein of a metal chealate (poly his) plasmid look like?
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
51
describe the process of purification using poly histidine tags
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
52
what are four general problems assoviated with the production of recombinant proteins in E coli?
* 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
53
how can we counter the general problem at the level of translation in the production of recombinant protein in e coli?
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
54
Frequency of the codons used is dependent upon...
...the abundance of the tRNAs in those cells
55
different codon bias in bacterial cells could result in what issue with the result recombinant protein?
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
56
how do we counter the problem of degredation in recombinant proteins produced in e coli?
can use fusion vectors and ION- strains of bacteria (those that are protease deficient)
57
how do we solve the problem of processing in recombinant protein production in e coli?
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
## Footnote
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
58
what are the most efficient hosts for human protein production?
higher eukaryotes
59
how are higher eukaryotic cells used in protein production?
multiple plates with a single layer of cells
60
what are the three kinds of promoters that can be used in higher eukaryotes?
* viral promoters - eg CMV or SV40 (strongest promoters we know and work in virtually any animal cell)
* heat shock promoter
* mouse metallothionein promoter
61
what are heat shock promoters induced by?
induced at 40 degrees celcius
62
how are mouse metallothionein promoters induced?
induced by zinc
63
what are two issues with expression in animal cells?
relatively low and hard to grow in bulk
64
what are the components of the expression vector for mammalian cells?
(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
65
what is the purpose of the signal sequence from immunoglobulin gene in the expression vector for mammalian cells?
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)
66
what is the purpose of the Myc epitope in the expression vector for mammalian cells?
to check for expression of the gene
67
what is the purpose of the 6x His tag in the expression vector for mammalian cells?
to allow us to purify our protein
68
what are the advantages and disadvantages of mammalian cells used for protein production?
- Best host for human proteins - expression generally good
- Produce the same post translational modifications
- Expression is generally low
- Hard to grow in bulk
69
what is Pharming?
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
## Footnote
In pharming, these genetically modified (transgenic) animals are used mostly to make human proteins that have medicinal value.
70
where are transgene most commonly expressed in pharming?
o Eggs of chickens
o Milk of cows, goats or sheep
Take eggs and milk on daily basis and take protein from that
71
when was the first transgenic farm animal?
in 1985
tracey a sheep expressing alpha 1 antitrypsin
72
what are the four steps to produce a transgenic animal?
1) Preparation of transgene
2) Pronuclear microinjection
3) Transfer to pseudopregnant female
4) Screen offspring for presence of transgene
73
give two examples of mammary gland specific promoters
casein or lactoglobulin – promoters only active in the mammary gland
74
in the preparation of the transgene for pharming, after the gene is cloned in what happens?
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
what are the advantages of having a linear piece of DNA?
more efficient at integrating into genome of host
76
describe pronuclear microinjection
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
what are the potential problems with pronuclear microinjection?
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
## Footnote
Expression lower than you want and you don’t find out until they reach maturity
78
what is the alternative to pronuclear microinjection that ensures high expression levels?
nuclear transfer
79
even though the DNA in the cells is the same why are neurons and muscle cells structually and functionally distinct?
due to different DNA methylation patterns
80
Low levels of methylation at the promoter means...
...RNA pol can bind and the gene is transcribed
81
High levels of methylation at the promoter...
....block RNA pol and transcription factors from binding so the gene is switched off
82
what is DNA methylation?
- Addition of methyl (CH3) groups to DNA
- Usually the C bases is methylated
83
describe the changes in methylation patterns that occur in very early life
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
describe why there are structural and functional differences between neurons and muscle cells?
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
what is the differnce between pluripotent and unipotent cells?
The only difference in the DNA is the level of methylation
86
when was dolly the sheep cloned?
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
describe cloning by the Dolly-Roslin technique
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
breifly describe why nuclear transfer has higher expression than pronuclear microinjection
* 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
what are theadvantages of the production of cloned animals which have originated from a single recombinant cell?
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
what expression system should be used if the protein of interest is unglycosylated?
easiest and cheapest is bacterial cells
91
what expression system should be used if the protein is glycosylated?
animal cells or animals themselves
92
how is insulin made in the body?
made as preproinsulin which is then folded and cleaved
the alpha and beta chains are held together by disulphide bridges
93
is insulin glycosylated or not?
insulin is not glycosylated
93
is insulin glycosylated or not?
insulin is not glycosylated
94
how has insulin be previously obtained and why were new methods needed?
- insulin was obtained from the pancreas of cows and pigs
- the side effects
## Footnote
The most common adverse reactions reported with this insulin include hypoglycemia, allergic reactions, injection site reactions, lipodystrophy, weight gain, and edema
95
describe the steps to producing synthetic insulin
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
what is factor VIII?
The F8 gene provides instructions for making a protein called coagulation factor VIII playing a central role in blood clotting
97
how is factor VIII previously produced?
purification from human blood
98
what is the problem with getting factor VIII from purified human blood?
99
give 3 features of factor VIII that make it difficult to make
* very large over 186kb
* contains 17 disulphide bridges
* only active after extensive glycosylation
100
is factor VIII glycosylated or not?
factor VIII is extensively glycosylated
| (only active after it is)
101
cell culture costs ____ per gram of protien
$100
102
pharming can cost ____ per gram
$2 - $20
103
what are the most efficient bioreactors?
cows
8000l of milk per yr with 40 to 80kg of protein
104
what is the most common hemophilia deficiency?
factor VIII
105
what animals are used to produce alpha 1 antitrypsin for treatment of emphysema?
sheep
106
what animals are used to produce CFTR for treatment of cystic fibrosis?
sheep
107
what animals are used to produce tissue plasminogen activator for treatment of thrombosis?
sheep
pig
108
what animals are used to produce factor VIII for treatment of hemophilia?
sheep
pig
cow
| sheep and pig also used to make factor XI
109
what animals are used to produce fibrinogen to treat wound healing?
sheep
cow
110
what animals are used to produce human protein c for treatment of thrombosis?
goat
111
what animals are used to produce antithombin 3 for treatment of thrombosis?
goat
112
what animals are used to produce glutamic acid decarboxylase for treatment of type 1 diabetes?
goat
113
what animals are used to produce Pro542 for treatment of HIV?
goat
114
what animals are used to produce alpha lactalbumin for anti infection?
cow
115
what animals are used to produce collagen 1 and 2 for tissue repair/ treatment of arthritis?
cow
116
what animals are used to produce collagen 1 and 2 for tissue repair/ treatment of arthritis?
cow
117
what is biosteel?
BioSteel™ Goats have been genetically modified to produce the protein from Golden Orb Weaver Spider (Nephila clavipes) silk in their milk
## Footnote
spider silk is stronger and more flexible then steel and offers a lightweight alternative to carbon fibre
118
what conditions could stem cells possibly treat?
* Spinal cord injuries
* Parkinsons disease,
* AD
* Type 1 diabetes
* CVD
* deafness
119
how do stem cells avoid senescence?
they express high levels of telomerase
120
describe the terms unipotent, multipotent, pluripotent and totipotent
- 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
when do cells in the embyro transition from totipotent to pluripotent?
From the zygote to the morula we would class the cells as totipotent
From the blastocyst, the inner cell mass cells are pluripotent
122
where do embryonic stem cells come from?
the inner cell mass from blastocysts left over from invitro fertilisation in the lab or aborted fetuses
123
what is the key regulator of potency?
Oct 4
124
where have adult stem cells been found?
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
give 4 features of Oct 4
| domain, binding location, expression, associate proteins
- 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
where do we find high levels of Oct 4 in the fertilised egg/embryo
we find high levels of Oct 4 in the zygote, morula and inner cell mass
## Footnote
when oct 4 levels decrease they allow the TE to form.
127
what happens to oct4 when cells begin to differentiate?
Oct4 will go down
128
what two jobs is Oct4 doing in stem cells?
activate self renewal genes and repress/silence developmental control genes
129
what are the two sources of embryonic stem cells?
1. Excess fertilised eggs from IVF clinics
2. Therapeutic cloning
130
what promoters of self renewal genes do Oct4, Nanog and Sox2 activate?
Oct4 Sox2 Nanog C-Myc Myst3
131
what promoters of developmental genes do Oct 4 Nanog and Sox2 silence?
Neurog1, Pax6, Gata4, Cdx2
132
can therapeutic cloning be used to produce stem cells?
what benefits and ethical considerations do they have?
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
what is the primary role of adult stem cells?
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
where are adult stem cell primarily isolated from?
bone marrow
(they are easier to activate to some cell types than others)
135
what do heamatopoietic stem cells differentiate into ?
all types of blood cells
136
what do mesenchymal stem cells differentiate into?
bone, cartilage and fat
137
what do umbilical cord stem cells differentiate into ?
blood bone and neurons
138
what do neural stem cells differentiate into ?
neurons, astrocytes and oligodendrocytes
139
compare embryonic stem cells with adult stem cells
| 3
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
when were induce plurpotent stem cells first developed?
in 2007
141
application of what four pluripotency factors are used to convert an adult cell back into an embryonic stem cell?
Oct4, Sox2, c-Myc , KLF4
142
what other combinations of plurpotency factors work to produced iPS cells?
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
what combination of plurpotency factors enhances the formation of iPS cells?
Oct4, nanog, sox2, lin28, cmyc and klf4
144
how much does combination of all 6 pluripotency factors enhance iPS fromation?
enhances iPS formation 10 fold and reduces time of reprogramming from 26 days to 17 days
145
what can influence the differentiation capacity of resultant iPS cells?
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
## Footnote
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
how are iPS cells created?
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
how can iPS cell production be used to correct genetic defects/ diseases caused by mutation?
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
what are the potential uses of iPS cells?
drug modelling, disease modelling, quality control for safety and efficacy (cell identity, purity, and potency) and cell based therapy
149
what are the pros of iPS cell technology?
Pros:
– Cells would be genetically identical to patient or donor of skin cells (no immune rejection!)
– Do not need to use an embryo
150
what are the cons to iPS cell technology?
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
describe the stem cell trials for cardiac disease
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
what was the outcome of the stem cell trials for cardiac disease in humans?
* 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
how can be stem cells be used to treat diabetes?
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
what is the molecular basis of diabetes?
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
how can we enhance the ability of stem cells to get to their required site of action?
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
what two ways can iPS cells be created/implanted for diabetes?
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
how does IPTG induce expression by the Lac promoter (operon)?
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
how much more efficient is the tac promoter?
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
what represses the Tac promoter?
how is expression controlled?
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
what is polyadenylation?
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.
## Footnote
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
what is factor Xa?
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
how is pGex vector protein purified?
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
what was the first vector of the type that helped increase the stability of the protein while helping purify it as well?
pGex vector
164
what is required for the recombinant protein to be secreted into milk?
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
whats needed for recombinant protein expression in the mammary gland? give three examples
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
why is the nuclear DNA injected into the male pornucleus in pronuclear microinjection?
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
