Transgenics

Question 1

When is transgenics used?

Answer 1

1) To study the function of the genes
2) To produce novel products
3) As model systems for human diseases or crop plants

Question 2

What is transgenics?

Answer 2

Organisms with transgenders

Transfer a gene into some organisms

Question 3

How are transgenic animals generated by microinjection of DNA into fertilized eggs?

Answer 3

1) DNA is injected into the nucleus of a zygote
2) The injected zygote is implanted into a pregnant female mouse
3) Each of the offspring is examined for the presence of the injected DNA to identify transgenic mice because very few zygotes actually take in this foreign DNA

Question 4

How are transgenic animals generated by embryonic stem cell technology?

Answer 4

1) Mate mice from a dark-coloured strain to obtain embryos at the blastocyst stage
2) Culture embryonic stem cells from the inner cell mass
3) Transfect the embryonic stem cells with marker DNA
4) Inject the transfected embryonic stem cells into a blastocyst from light coloured parents
5) Implant the injected blastocyst into a light-coloured female to obtain a chimeric off-spring
6) Mate the chimera to a light coloured mouse to obtain off-spring
7) Examine the DNA from the dark coloured offspring to determine if they contain marker DNA sequences. Mice that have these marker DNA sequences are transgenic

Question 5

What are chimeric animals?

Answer 5

Progenies of pseudopregnant females are chimeras, because these animals have two types of cells that have contributed to the formation of adult tissues, some with the transgene and some without

Question 6

How are true transgenic animals obtained?

Answer 6

True transgenic animals may be obtained through breeding chimeras, only if some germ line cells in the chimeras have the transgene

Question 7

Explain the chimeric mouse

Answer 7

A chimeric mouse is a mosaic: cells developed from the original mouse, but also the engineered stem cells
cells from both randomly integrate, and you hope that the engineered stem cells integrate into the germ-line.
There are cells from both the white, and black (engineered stem cells) mouse
Only dark coloured mice are possibly transgenic
Light coloured mice are not transgenic

Question 8

Why are only dark coloured mice possibly transgenic?

Answer 8

White mouse genotype: (bl-/bl-, trans-/ trans-)
Chimeric genotype: (bl+/bl+, trans+/trans-) if genetically engineered embryonic stem cells from black mice
When you cross the white mouse with the genetically engineered mouse you can get:
–> (bl+/bl-, trans+/trans-) OR (bl+/bl-, trans-, trans-): Mice are all grey here, but only some have the transgene
The chimeric genotype may have germ cells that have the genotype: (bl-/bl-, trans-/trans-), so when you cross this with the white mouse you get:
(bl-/bl-, trans-/trans-): Mice are white with no transgene

Question 9

What does it mean to knock out and knock in a gene?

Answer 9

Knock out: targeted gene deletion, remove a gene
Knock in: targeted gene replacement, swap in and swap out a gene
Targeted: control for where the gene goes

Question 10

How can a gene be disrupted by targeted homologous recombination?

Answer 10

There is a chromosome that contains a p and pa arm, which are homologous arms
There is a target vector which contains an arm that is homologous to the p and an arm that is homologous to the pa, but the target vector also contains a selectable marker gene.
1) The p and pa arm in the chromosome undergo homologous recombination with the target vector (p and p) and (pa and pa).
2) The resulting chromosome contains the p and pa arm, but also contains the selectable marker gene from the target vector, which is in between the p and pa

Question 11

How does positive-negative selection for targeted integration occur?

Answer 11

There are two ways:

1) Non-specific integration
2) Homologous recombination

Question 12

Explain how the non-specific integration for positive-negative selection for targeted integration occur?

Answer 12

There are thymidine kinases (tk1, tk2), two DNA sequences (HB1 and HB2) that are homologous to a specific chromosomal region in the recipient cell, Neo r, which confers resistance to the cytotoxic compound G-418, and the transgene.

1) This vector is incorporated into the chromosome.
2) After incorporation, cells are selected for resistance to G-418 and glanciclovir. –> Glanciclovir is cytotoxic to cells that synthesize thymidine kinases.
3) Other non-homologous integrations (or non-specific integration) can occur and produce inserts with one or the other of the tk
4) After treatment with G-418 and ganciclovir, the cells with non-specific integration, that includes at least one tk are killed
5) Cells do not carry the thymidine kinase, because any cells that carry the tk will be killed by glanciclovir.
6) Final cells do not have the tk, but have everything else

Question 13

Explain how the homologous recombination for positive-negative selection for targeted integration occur?

Answer 13

1) The HB1 and HB2 in the vector, as well as the HB1 and HB2 in the chromosome undergo homologous recombination.
2) The tk1 and tk2 do not undergo homologous recombination with the chromosome, and as such are not integrated at the targeted site.
3) Cells survive double selection because tk is not present, so cells will not be killed by glanciclovir

Question 14

How does genetic modification with the Cre-loxP recombination system work?

Answer 14

Cre: enzyme
loxP: DNA
The Cre-loxP recombination system is derived from genetic elements of the bacteriophage P1
A loxP site consists of two 13 bp inverted repeats that are separated from each other by an 8 bp spacer sequence
Two remote loxP sites come together, each of which is cleaved by the Are recombinase within the spacer regions between the repeat sequences; the two DNAs exchange and join to form recombined DNA molecules

Question 15

Can a loxP site have repeats in the same orientation?

Answer 15

The loxP site can have repeats that are inverted, separated by a spacer or have repeats that are in the same orientation, also separated by a spacer

Question 16

What happens if the two loxP sites have an opposite direction?

Answer 16

The Cre recombinase inverts the sequence in between the two opposite loxP sites. For example, if the sequence in between goes in the right direction, it is inverted and now goes in the left direction through looping

Question 17

What happens if the two loxP sites have the same direction?

Answer 17

The Cre recombinase excises or removes the sequence in between the two loxP sites facing the same direction through a looping mechanism

Question 18

How is the Cre-loxP site used to inactivate a gene?

Answer 18

Cre enzyme is only expressed in certain cells, so this is a tissue specific knockout system

1) Let’s say the loxP sites have the same direction, and exon 2 is in between these two loxP sites that have the same direction,
2) The Cre recombinase comes in, and the two loxP sites line up, and exon 2 (the sequence in between the two loxP sites) is removed/degraded.
3) The resulting product is one loxP site in between exon 1 and exon 3, with exon 2 being removed, leading to the inactivated gene
4) The degraded product is exon 2, with one loxP site

Question 19

How can the Cre-loxP system be used to activate a transgene?

Answer 19

Normally the promoter is blocked, so the gene is turned off
There are two loxP sites that have the same direction. The promoter that is blocked, is in between these two loxP sites which have the same direction
1) The Cre recombinase comes in, which allows the two loxP sites to line up, which allows for the blocked promoter to removed
2) The final product contains the three exons, the blocked promoter is removed, but there is one loxP site before exon 1 which results in an activated gene
3) The degraded product is the blocked promoter, with one loxP site

Question 20

What is the tetracycline inducible system?

Answer 20

The Tetracycine receptor (TET) is a bacterial sequence specific DNA binding protein.
Its binding to the promoter of the tetR gene is regulated by the presence of Tetracycline

Question 21

How does the tetracycline inducible system work?

Answer 21

In the tetracycline inducible systems the TET DNA binding domain (DBD) is fused to a mammalian transactivation domain (TA)
The synthetic tetracycline responsive promoters contain the TET-DNA binding site from the tet R promoter (TRE) inserted upstream of basal mammalian promoter elements
The TA is the activator, which binds to the DBD which is the DNA binding domain which binds to the TRE on the DNA

Question 22

What is the TET-off inducible gene expression system?

Answer 22

The TET-off protein is a mutant, which can not bind DNA in the presence of tetracycline
Upon the removal of tetracycline TET-off binds DNA

Question 23

What is special about the TET-off inducible gene expression system?

Answer 23

Expression plasmids are designed to respond to TET-off-TA
The TET-off system works well in tissue cultured cells, but not so well in whole organisms because:
Side effects of continuous usage of tetracycline
Degradation of tetracycline (multi-drug resistance)

Question 24

How does the TET-off inducible gene expression system work?

Answer 24

The system contains two plasmid: one expresses the TET-off-TA fusion protein and the other contains the protein encoding sequence under the control of the TET-responsive promoter
In the presence of tetracycline: the TET-DBD and TA cannot bind to TRE, and no gene activation
In the absence of tetracycline: the TET-DBD and TA bind to the TRE, which causes gene activation

Question 25

What is the TET-on inducible gene expression system?

Answer 25

An alternative TET system, which works equally well in tissue culture and in organisms.
TET-on cannot bind DNA in the absence of tetracycline, but upon the addition of tetracycline the TET domain binds DNA and activates the promoters

Question 26

How does the TET-on inducible gene expression system work?

Answer 26

In the presence of tetracycline: the TET-DBD and TA binds TRE, and gene activation
In the absence of tetracycline: the TET-DBD and TA cannot bind to the TRE, which causes no gene activation

Question 27

What does it mean when the TET-off and TET-on systems are leaky?

Answer 27

Low levels of expression without stimulation of the promoters
This can lead to:
Toxic proteins
High background effects

Question 28

What happens in the absence of Doxycycline?

Answer 28

The binding of the inhibitory factor to the tetO region is greater than the activating/permissive factor, silencing gene expression

Question 29

What happens in the presence of Doxycycline?

Answer 29

High level of gene expression

Question 30

What is CRISPR-Cas9 Genome Editing System?

Answer 30

CRISPR- Cas is derived from a prokaryotic adaptive immune system
It contains two biological components: the RNA guided DNA endonuclease Cas9 and a chimeric single guide RNA (sgRNA)
sgRNA consists of a CRISPR RNA component and a trans-activating crRNA component, which binds to Cas9 and directs it to a genomic sequence of interest via base pairing to the target sequence

Question 31

How does the CRISPR-Cas Genome Editing System work?

Answer 31

CRISPR-Cas may be programmed to cut a DNA sequence of interest simply by altering the 20 nt variable spacer sequence of the crRNA
The only sequence constrain is the presence of a PAM sequence immediately proximal to the region of crRNA homology on the DNA target.
Other Cas9 proteins have different sites

Question 32

What are the important sequence in CRISPR-Cas Genome Editing System?

Answer 32

1) DNA target
2) Cut sites
3) PAM: Need the PAM sequence and is often close to the target DNA sequence, and the PAM sequence needed to be located before targeting can occur
4) The crRNA is the 20 nt targeting specificity
5) The tracrRNA is the constant sequence
6) There is a sgRNA linker

Question 33

How does genome editing with CRISPR-Cas9 work?

Answer 33

1) The resulted DNA double stranded breaks can be repaired via the non-homologous end joining pathway, which is error-prone, insertions or deletion mutation can occur
2) In the presence of an exogenous donor DNA template, the DSB can be repaired via the homology-directed repair pathway, which can be used for engineering precise DNA modifications

Question 34

How does the HDR pathway work?

Answer 34

1) The DNA has a cleaved site, and the donor template can be inserted into where the cleaved site was.

Question 35

How can CRISPR-Cas be used to pool genetic screens?

Answer 35

Reagents are usually synthesized and constructed a pool, but viral transduction limits the transgene copy number
The viral integration enables readout through PCR and next-generation sequencing, which is based on the comparison of the abundance of the different genetically integrated transgene reagents between samples
You introduce pool of mutants and then genetic screen

Question 36

How are pooled genetic screens performed using plasmids?

Answer 36

1) Synthesize DNA corresponding to RNA
2) Pooled plasmid cloning: each plasmid transcribes the RNA to be used
3) Virus production and transduce virus into cells
4) Select for transduced cells: Positive selection, control, negative selection
5) PCR genomic DNA
6) Next generation sequencing
7) Matrix of sgRNA abundances

Question 37

How are pooled genetic screens performed using sgRNA?

Answer 37

1) Pooled sgRNA oligos
2) Pooled sgRNA cloning
3) sgRNA library
4) Lentiviral packaging: Lentiviral infection
5) Selection: +Treatment, - Treatment
6) Deep sequencing of sgRNA barcodes

Question 38

What is deep sequencing?

Answer 38

Sequencing a genomic region many times (100s or 1000s times) using NGS approach to detect rare clonal types, cells, or microbes within a biology sample