Engineering the Genome

Question 1

What is bacterial transformation and how does it confer an advantage to bacteria?

Answer 1

Transformation is when bacteria transport exogenous DNA into their cell, and incorporate it into their own genome. Bacteria that are able to do this are termed competent. It confers an advantage as it allows them to increase genetic diversity - binary fission produces genetically identical cells.

Question 2

Explain the concept of competency and how it works naturally and artifically

Answer 2

Naturally competent bacteria can take up exogenous DNA but not circular plasmids where artificially competent bacteria can. For naturally competent bacteria, the DNA that they take up must be closely related - process called conjugation (uses HR)

Question 3

Detail the process of how naturally competent bacteria take in exogenous DNA

Answer 3

DNA binding proteins on the surface of the cell bind the DNA, and it is turned into ssDNA by a nuclease as it enters the cell. RecA proteins use HR to integrate it into the plasmid.

Question 4

What is gene targeting?

Answer 4

The design and delivery of DNA into a cell in order to make a defined genomic modification via Homologous Recombination

Question 5

How can we use gene targeting in bacteria and simple eukaryotes to modify a specific fragment of DNA?

Answer 5

A linearised plasmid (Targeting construct) which contains homologous regions flanking a selectable marker gene is introduced to the chromosome. The homologous regions align, and undergo HR. This introduces the marker gene into the chromosome. If the marker gene interrupts a gene, the effect of the disruption can be studied. This is reverse genetics

Question 6

How can we use gene targeting in bacteria and simple eukaryotes to modify a single base in the DNA?

Answer 6

A circularised plasmid (Insertion construct) which contains two homologous regions which have a single nucleotide modification is introduced to the chromosome. HR between first homologous region incorporates the whole plasmid into the chromosome, and the plasmid is duplicated, along with the second region of homology and the selectable marker. The second homologous region crosses over with the original chromosome, which makes the plasmid excise itself, leaving the single nucleotide modification behind

Question 7

Why do higher eukaryotes lack competent cells?

Answer 7

Higher eukaryotes introduce genetic variation by promoting HR between chromosome homologues in meiosis, so competency is not needed

Question 8

What is transfection and transformation in terms of mammalian cells?

Answer 8

Transfection is the artificial introduction of DNA to mamallian cells and transformation refers to the acquisition of malignant characteristics

Question 9

Describe the transfection method of Viral Transfection

Answer 9

The DNA is packaged into a viral particle which delivers the DNA to the cell. This is efficient but the creation of a viral particle is time consuming

Question 10

Describe the transfection method of Electroporation

Answer 10

A capacitor is discharged through a mixture of cells and DNA. This induces transient membrane pores through which the DNA can enter the cell. This is effective for many different types of cell but requires specialist equipment

Question 11

Describe the transfection method of Microinjection

Answer 11

The DNA is injected directly into the nucleus with a microneedle. This method is efficient but time consuming and requires much skill

Question 12

Describe the transfection method of Lipofection

Answer 12

Cationic (positively charged) lipids are complexed with negatively charged DNA and endocytosed by the cells. This method is simple but not very useful for the majority of cells

Question 13

By which process is the DNA taken up by cells incorporated into the genome?

Answer 13

The process by which the DNA integrates is NHEJ, due to the fact that for HR the inserted DNA needs to be homologous and the fact that HR is switched off in G1. Also, NHEJ is error prone meaning it often results in the integration of DNA. Due to the integration method being NHEJ, the DNA is randomly integrated into the genome

Question 14

How are marker genes useful when present in the DNA construct?

Answer 14

They allow for the selection of stably transfected cells in antibiotic

Question 15

How do reporter genes allow us to measure the effect of genetic modification on a particular signalling pathway?

Answer 15

A reporter gene (GFP) is linked to transcriptional control elements from a gene induced by the pathway of interest. The TFs will act on the gene which codes for them (cis acting) so the expression levels of the gene will correlate to the fluorescence level and that will tell us about the activity of the pathway of interest

Question 16

What are chromosome position effects?

Answer 16

When the chromatin features close to the site of integration interfere with the expression of the gene

Question 17

How do we overcome chromosome position effects?

Answer 17

We target the transgene into a housekeeping gene locus so that we know the gene will be constitutively expressed. This produces a stably transfected cell line

Question 18

Why are stably transfected cell lines neccessary?

Answer 18

For pharming - eg. producing human blood clotting factors in milk, antibodies, etc. We use mammalian, preferably human, cell lines to do this so that we can ensure the proteins have the appropriate post transcriptional modifications for them to function correctly in humans

Question 19

Describe how pronuclear injection is used to create transgenic animals

Answer 19

One of the pronuclei in the zygote are injected with DNA which randomly integrates into the genome. The injected zygote is transplanted into a foster mother and the pups are screened for the transgene. Integration by HR is too inefficient for this technique to be used for gene targeting, until customised nucleases became common. The transgenes are subject to chromosome position effects

Question 20

Describe how Embryonic Stem Cells are used to create transgenic animals

Answer 20

ESCs are isolated from an embryo and can be grown indefinitely in culture. They can differentiate into any cell type but inhibitors in the media don’t allow this. ESCs are particularly proficient at HR so targeted:random integration is 1:10. These ESCs are then introduced into a host blastocyst which is implanted into the mother. The resulting offspring are then chimeric, some cells derived from ESC, some from blastocyst. If germ cells are ESC derived, offspring will be heterozygous for the modification. Sibling mating -> homozygous for the modification.

Question 21

What are transgenic animals created by ESCs used for?

Answer 21

Creating KO animals to assess the functions of genes and other genomic features
Studying genomic regions controlling complex physiology, such as the brain, where a cell line is not informative
Create mutations that mimic human disease symptoms in order to create a human disease model in the animal
Allow the monitoring of gene expression by using a reporter in the gene

Question 22

How is Somatic Cell Nuclear Transfer used to create transgenic animals?

Answer 22

A somatic cell nucleus is transplantedi into an enucleated egg which is then transplanted into a foster mother. This allows the creation of transgenic animals by modifying the DNA in the nucleus of the somatic cell. Allows for both random and targeted integration, and is more efficient than Pronuclear Injection.

Question 23

What are transgenic animals created by SCNT used for?

Answer 23

Transgenic animals are used to improve food production, such as genes for growth. Also used for pharming - blood clotting factors in cow milk. Also can eliminate mad cow disease. Also being used to create transgenic pigs without antigens responsible for rejection in xenotransplantation

Question 24

Explain the idea of conditional gene targeting

Answer 24

Conditional gene targeting is used when we need to investigate the effect of a gene disruption in a specific tissue. Also, we can use it if we want to see the effects of the disruption at a later stage in development. It can help us to avoid embryonic lethality too

Question 25

Explain the LoxP/Cre Recombinase and how it allows us to perform conditional gene targeting

Answer 25

In the responder animal, the gene of interest is “floxed” by LoxP sites. In the regulator animal, the Cre Recombinase is put under the control of a strong promotor appropriate for the needs of the experiment, such as a tissue specific promotor. This happens in the regulator animal. Then offspring of the responder and the regulator animal will then have both the Cre recombinase and the floxed gene and will excise its own gene out. Also, careful positioning of the LoxP sites can facilitate translocations

Question 26

Explain the CRISPR-Cas system as it naturally occurs in bacteria

Answer 26

CRISPR are gRNA regions from previous viral infections that the bacteria store for re encounters. Upon reinfection, the CRISPR guides the Cas protein to the virus where it cuts the DNA.

Question 27

How can CRISPR-Cas9 be used to help humans

Answer 27

We can make our own gRNAs to guide the Cas proteins to a desired sequence in the genome where the Cas protein will cut both strands. This is called gene editing. The nuclease is often caused to be expressed in cells by an expression plasmid or a viral vector. Or the purified Cas9-gRNA complex can be delivered directly to the cell

Question 28

What role does the PAM sequence play in the CRISPR-Cas9 system?

Answer 28

The gRNA must be followed by a PAM sequence which is 3 nucleotides 5’-NGG in this case. It allows the gRNA to bind the section of DNA but it doesn’t hybridise. Each different type of Cas9 protein can recognise a different PAM sequence.

Question 29

How else can CRISPR-Cas9 systems be used to help humans?

Answer 29

The Cas can be a nickase by deactivating an active site. Deactivating both active sites will make dead Cas9 which can be used as a target-specific DNA binding protein. Also, we can fuse fluorescent proteins to the Cas9 to visualise the position of the DNA in the cell. Also, deaminases can be fused to the dead Cas9 to turn C to T to make point mutations. Also, the dead Cas9 can be fused with transcriptional activators to transcribe certain genes, the same for silencing.

Question 30

How can CRISPR systems be used to create Knock outs and Knock ins?

Answer 30

NHEJ CRISPR creates indels which results in KO cells. In the presence of a template, we can perform knock ins by HR with dsDNA templates and for ssDNA templates it results in 1 or a few nucleotide modification. A successful knock in will result in an indel of the other allele so we need to screen for the appropriate genotype. NHEJ is still active when a tempalte is present - need inhibitors