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GENE 9: Engineering the genome Flashcards

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1
Q

How is exogenous DNA incorporated into the bacteria’s genome?

A

Homologous recombination, if they have homology to the host chromosome and their replication origins are inactivated

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2
Q

Bacteria capable of taking up DNA are known as?

A

Competent

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3
Q

Naturally competent bacteria do not usually take up circular plasmids, how can they be made to do so?

A

They can be made artificially

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4
Q

What does the RecA protein do?

A

Uptakes ssDNA

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5
Q

How does transforming DNA get into the bacterial cell?

A

Binds to DNA binding, then competence-specif single-stranded DNA binding proteins bind to the DNA pulling it in.

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6
Q

What is the advantage of having a system to recombine DNA into their genome?

A

Increases genetic diversity

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7
Q

How do bacteria produce asexually?

A

By binary fission, where two progeny cells are genetically identical (except in the case of rare mutations)

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8
Q

What is gene targeting?

A

Transforming bacteria with defined pieces of DNA so as to make defined modifications to the bacterial genome via HR

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9
Q

What can be used as a possible way to modify genomic loci of unknown function?

A

selected marker genes

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10
Q

How does gene targeting with a replacement construct work?

A

A linearised plasmid carries regions of homology with the chromosome, separated by a gene marker. Other regions of the plasmid are unnecessary for this type of gene targeting and are ideally removed. After delivery into cells, the construct aligns with its homologous regions in the chromosome and undergoes HR involving 2 crossovers. The resulting replacement event introduces the marker gene between regions in the chromosome. Cells modified in this way are selected in antibiotic. If the near regions are known to carry a gene, the effect on cells of disrupting it in this way can be studied.

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11
Q

How do you create a single nucleotide change (simple)?

A

Using a targeting plasmid known as an insertion construct

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12
Q

How do you create a single nucleotide change (detailed)?

A

Step 1: a single crossover between one homologous region which results in insertion of the entire plasmid into the target locus and duplicate copies of the homology region. Cells that have undergone this event will express the antibiotic resistance gene

Step 2: Involves a second crossover on the other side of the mutation. This results in excision of the insertion construct, which now lacks the mutation. The original target locus is left modified by the subtle mutation

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13
Q

T/F: Linear double stranded DNA is converted to single-stranded DNA during natural bacterial transformation?

A

True

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14
Q

The selectable marker is positioned outside the homology for insertion constructs or replacement constructs?

A

insertion constructs

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15
Q

The selectable marker is positioned inside the homology for insertion constructs or replacement constructs?

A

replacement constructs

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16
Q

Can insertion constructs or replacement constructs inactivate a target gene without leaving a sequence duplication?

A

replacement construct

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17
Q

How do higher eukaryotes generate genetic diversity?

A

By promoting HR between chromosome homologues during meiosis, there is no need for mammalian cells to take up exogenous DNA as bacteria do

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18
Q

What is transfection

A

Artificially introducing plasmids to mammalian cells

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19
Q

Name the types of common transfection methods

A

Viral transfection
Microinjection
Lipofection
Electroporation

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20
Q

Explain viral transfection delivery, with an advantage and disadvantage

A

DNA is packaged into viral particles that deliver DNA into target cells.
A: efficient delivery
D: Time consuming to make particles

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21
Q

Explain microinjection and an advantage and disadvantage

A

Direct injection of DNA into the nucleus using a micro-needle.
A: Efficient
D: Needs much time and skill

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22
Q

Explain microinjection and an advantage and disadvantage

A

A capacitor is discharged through mixed cells and DNA inducing transient membrane pores for DNA uptake
A: Useful for large cell numbers
D: Requires specialist equipment

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23
Q

Explain Lipofection and an advantage and disadvantage

A

Complexes between cationic lipids and negatively charged DNA are endocytosed by cells
A: Simple
D: Less useful for large cell numbers

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24
Q

In what way do mammalian cells insert exogenous DNA into chromosomes?

A

Unlike bacteria and yeast, randomly, with no requirement for sequence homology to the recipient genome.

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25
What is the mechanism that underlies random integration and why it is preferred over targeted integration?
Non-homologous end joining It is error prone: NHEJ errors include not only the formation of chromosomal translocations but also the incorporation of exogenous DNA at double strand breaks, and NHEJ is active throughout interphase.
26
What’s wrong with targeted integration using HR?
HR is inactive during G1 | Integration of exogenous DNA by HR, by definition, can only occur at the homologous chromosomal site
27
What are the two types of integration?
Random | Targeted
28
What way can you screen for rare targeted integrants among a majority of clones that are random
A reporter gene, such the GFP gene, is linked to cis-acting transcriptional controlelements from a gene induced by a signalling pathway of interest. After stable integration of theresulting plasmid into a suitable cell line, the effects of various molecules or growth conditions on thesignalling pathway can be conveniently assessed by measuring the amount of green fluorescence.
29
What are stably transfected cell lines used for in commercial production?
Pharmaceuticallyimportant proteins such as blood clotting factors and antibodies
30
What advantage does using human (mammalian) cell lines as opposed to bacteria or yeast have the advantage of?
Proteins are made with theappropriate post-translational modifications, such as glycosylation, required for full protein function inpatients.
31
How do you optimise protein production of inserted genes?
The genes for such proteins are usually cloned into a vector next to a strong viral promoter
32
When transfecting a gene into mammalian cell lines, what are the different methods by which you can do this? (list)
1) viral transfection 2) microinjection 3) lipofaction 4) electroporation
33
In what order are the steps to make an edited gene?
1) Anneal oligonucleotides 2) linearisae plasmids 3) ligate plasmids and oligonucleotides 4) transform bacteria with them (acquiration of characteristics) 5) transfect into HEK293/water cells (aquiration of characteristics too but in mannalian cells transformation means malignant) 6) test presence
34
When transfecting genes, does it need to be done in a targeted or random approach?
it is often sufficient for the transfected gene ('transgene') to be inserted at a random chromosomal site
35
Why does the efficiency of randomly integrated genes vary from site to site? Why is this so? What does this mean for finding a clone with the appropriate expression?
Due to !chromosomal position effects!. These effects reflect the chromatin status of the integration site. In practice it is therefore necessary to screen several or many clones before one with appropriate expression level is obtained
36
How can relaible and reproducible expression of transgenes be induced?
The transgene can be targeted by HR into a 'safe harbour' chromosomal site, i.e. a locus such as a house keeping gene, that is known to be permissive for transgene expression.
37
T or F? why? | Mammalian cells normally take up extracellular naked DNA
False | - mammalian cells can undergo HR and thats how they gain their genetic variation
38
T or F? | Transfected DNA integrates into host genomes mostly by HR.
False | its by NHEJ mostly
39
How can these transfection methods be used to introduce a genetic modification into every cell of an animal, including its germ cells?
Pronuclear injections Embryonic cell manipulation Somatic cell nuclear transfer
40
How is a pronuclear injection carried out?
Transgenic mice were made by microinjecting DNA into one of the two pronuclei of a zygote where it randomly integrates. Injected zygotes are then implanted into foster mothers and resulting pups are screened for presence of the transgene.
41
Before the development of customised nucleases why was integration by HR not a very useful method for gene targeting?
Its a very inefficient method
42
What are embroyonic stem cells?
Stem cells isolated from an early embryo and can be grown indefinitely in culture.
43
How can you get stem cells to grow into the type of cell you want them to be?
Although they have the potential to differentiate into any cell type, an inhibitory factor added to the culture medium prevents this. Cultured ESC are amenable to genetic manipulations including gene targeting.
44
Why are ratios of ESC, targeted to random integration, often quite high (e.g. 1 in 10 drug-selected cells)?
ESCs seem to be particularly proficient at integrating DNA by HR
45
How does transfection into embryonic stem cells work? What does this result in?
Targeted ESCs are introduced into a host blastocyst that is then implanted into a foster mother. The resulting offspring are chimeric: i.e. some of their cells are derived from the host blastocyst and some from the modified ESCs. If the germ cells of chimaeras are ESC-derived, they can be use for breeding to produce offspring whose every cell is heterozygous for the genetic modification. Sibling mating then generates animals homozygous for the genetic modification.
46
What are the 9 steps required for gene targeting in mice using Embryonic Stem Cells transfection?
1) Isolate blastocyte (strain 129) 2) Establish ES cells 3) Electroporate with targeting construct 4) Identiy and expand targeted clone 5) Inject targeted ES cell into host blastocyst (strain B6) 6) Inplant in foster mother 7) Look for chimaeric pups (based on coat colour) 8) Mate chimera and PCR check for +/- progeny 9) Sib-mate to homozygosity
47
List the possible uses of transgenesis using ESC in mice
1) Generating KO 2) studying complex body systems 3) Studying human disease 4) Studying gene expression
48
What are KO models?
Modifications that disrupt or remove a target region (knockouts) are useful for assessing the function or genes or other genomic features.
49
How can transgenesis using ESC be used for studying complex body systems?
Transgenic mice are particularly useful for analysing genomic regions controlling complex physiology (e.g. brain functions) that cannot be studied in cultured cells.
50
How has transgenesis using ESC been used for studying human diseases?
Subtle mutations that mirror human disease-causing mutations have been introduced into mice to generate animal model of human genetic disease (e.g. cystic fibrosis mouse).
51
How can transgenesis using ESC be used for studying gene expression?
Reporters, such as the GFP gene, can be inserted into a target gene to monitor its expression in live animals.
52
Outline the process of Somatic ell nuclear transfer?
An enucleated oocyte receiving the nucleus of a somatic cell and introduced into a foster mother can develop to term. By genetically modifying a somatic cell before transferring its nucleus to an oocyte, SCNT can be used for transgenesis.
53
What are the benefits SCNT more efficient than pronuclear injection?
This procedure is more efficient than standard pronuclear injection and can also be used for both random and targeted integration. An example of using the latter approach to generate a pig model of cystic fibrosis.
54
What are the steps used to generate a pig model of CF by gene targeting and SCNT?
The CFTR gene in primary pig fibroblasts was mutated by gene targeting, and their nuclei injected into enucleated oocytes which were then implanted into a foster sow. The resulting piglets were heterozygous for the CFTR mutation
55
What is SCNT used for?
To engineer livestock for improved food production and as living bioreactors for the production of protein pharmaceuticals ("pharming")
56
Give examples of SCNT being used to engineer livestock?
- Animals with transgenes to improve growth or meat composition or to produce proteins, such as human blood clotting factors, in their milk. - Cattle have also been developed in which the PrP gene has be knocked out to prevent transmission of mad cow disease. - Pig-to-human organ xenotransplantation, SCNT is also being used to generate knockout pigs lacking key antigens that promote rejection.
57
Name a germline problem
When a gene is edited this early on it can cause development problems and result in death in utero
58
How do you overcome the problems of - developmental problems - animals dying in utero when an important gene is KD in every cell - being unable to study the gene at later stages of development?
Conditional gene targeting has been developed
59
What system do many conditional gene targeting system?
Cre/lox
60
What is cre recombinase?
Its a bacteriophage protein
61
What does cre recombinase do?
It catalyses cre recombination between two copies of a specific DNA sequence (34bp sequences called LoxP)
62
How does cre recombinase work?
Finds LoxP sites cleaves the DNA then recombines excision the 34 bp portion of LoxP in between
63
Where cells can LoxP be used in?
Mammalian cells eg. mice
64
How can the Cre/Lox system be used to gene target in mice
Using gene targeting to introduce loxP sites around a target gene - performed in a responder animal
65
When is the cre/lox system used?
At a specific time in development or in a specific tissue
66
In what animal is the the genome modified to express cre recombinase? why is this done?
Regulator mouse | So it is inserted under the control of a specific promoter or inducer to meet the needs of the experiment
67
How do you obtain offspring with both the cre gene under the specific promoter and the target gene flanked with loxP sites?
Mating of the regulator and responder mice
68
What is the gene called if it is flanked by loxP sites?
Floxed
69
Where is the floxed gene knocked out?
Only in specific tissues and not all other cells
70
What does the different orientation and location of the LoxP sites mean for the cre/lox system?
Other modifications
71
If on two different chromosomes the loxP recombination results in translocation rather than excision, what does this mean for the experiment?
the location of the loxP sites is carefully designed for each experiment.
72
What transgenic method is used only on mice
ESC
73
What transgenic method is widely used in livestock
PI | SCNT
74
What transgenic method is useful only for random integration?
PI
75
What transgenic method requires DNA transfection in cultures cells?
ESC | SCNT
76
What transgenic method requires a host blastocyst?
ESC
77
How many times greater is gene targeting simulated when an endonuclease is used to cleave the target locus?
1000x
78
What are CRISPRs?
These are natural guide RNA (gRNA) regions of bacterial genomes that store bacteriophage DNA as a marker of prior infections
79
If a repeat bacteriophage infection occurs, what is expressed and guided to the viral genome by the gRNA?
An endonuclease (e.g. Cas9 in S. pyogenes)
80
What does Cas9 do?
It can cleave the virus and clear the infection
81
When crispr editing a gene what do you need to do to the gRNA?
reprogramme it for chosen target sequence
82
How does the guide RNA recognise te target strand
The guide RNA recognises its target DNA by standard base pairing rules, using just 20 nucleotides at its 5' end. The target sequence must be followed by the sequence NGG (N=any nucleotide) - the protospacer-associated motif (PAM).
83
What is the PAM recognised by?
Cas9/gRNA complex but does not hybridise to the gRNA
84
Once bound to its target sequence what does the Cas9/gRNA do?
It cleaves each strand of the DNA, 3 nucleotides upstream of the PAM, using two active sites in the Cas9 protein. CRISPR systems from bacteria other than S. pyogenes can be used similarly but recognise different PAM sites.
85
What are the steps used to create a pig model of Cystic fibrosis?
1) extract DNA from fetal fibroblast 2) introduce a null targeting vector 3) identify the targeted cells 4) use nuclear transfer to introduce vector into oocyte. The oocyte has had its genetic material removed 4) fusion/injection and activation of vector in oocyte 5) NT embryo is put into surrogate cow 6) cloned CFTR null heterozygote piglets produced
86
The use of customised nucleases to make targeted genome modifications is often referred to as?
gene editing
87
Where are nucleases expressed?
In cells from expression plasmids or viral vectors
88
What are the two strategies for customised nucleases in genome engineering?
1) genome expresses the endonuclease 2) No template 3) NHEJ 4) indel 5) Knockout 1b) template 2bi) dsDNA construct 2bii) ssDNA oligo 3b) HR 4) defined modifications 5) knock-outs and Knock-ins
89
Describe the two approaches for using customised nucleases in genome engineering
In the first approach, nucleases are used without an accompanying DNA. DSBs are therefore repaired by error-prone NHEJ giving rise to indels at the cleavage site. Most indels will disrupt gene expression if introduced into coding sequences. This approach therefore provides a simpler way to make gene knock-outs than standard gene targeting. Furthermore, this approach works so well in oocytes that multiple target loci can be inactivated simultaneously by injection of multiple nucleases in the same oocyte. This approach is now simpler and more efficient than the previous standard route for making knockout mice using ESCs. In the second approach, a template DNA for making a defined modification by HR with the target locus (a 'knock-in') is co-delivered with the nuclease. Frequencies of HR at the target locus are sufficiently high that the use of selectable marker genes is often unnecessary. Single-stranded oligonucleotide templates of 60-100 nucleotides can therefore be used to introduce modifications of one or few nucleotides. This saves time and expense of making targeting constructs.
90
Why is it still necessary to screen clones for the desired genotype following use of customised nucleases in genome engineering?
Even in the presence of an HR template, double strand break repair by NHEJ still occurs. Successful knock-in of one allele therefore is often accompanied by an indel in another target allele, either in the same cell or in other cells of the transfected population.
91
With CRISPR nucleases can gene knockouts can be made without targeting constructs?
yes
92
T or F | In S. pyogenes the PAM sequence is 5'-NGG.
True
93
Is the PAM is recognised by the gRNA?
No, Cas9/gRNA complex
94
What patients could in principle benefit from gene therapy? | i.e. the expression of a transgene in appropriate patient somatic cells.
Patients with inherited diseases (e.g. haemophilia or β-thalassemia) where a particular protein (e. g. clotting factor or β-globin) is defective or absent.
95
How would you ensure lasting benefit of the gene therapy?
The recipient cells must be - long-lived (e.g. liver, retina) - or somatic stem cells (e.g. haematopoietic stem cells (HSCs)) that can replenish tissues with high cell turnover (e.g. blood)
96
Ho wmay you overcome the problem of immune response to viral vectors?
Immunosuppression

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