7 - Gene Manipulation II

Question 1

What are the three broad steps of forward genetics (phenotype driven mutagenesis)?

Answer 1

1. Identification of heritable phenotype
2. Mapping and positional cloning
3. Discovering of causative gene

Question 2

What are the three broad steps of reverse genetics (gene-driven mutagenesis)?

Answer 2

1. Gene targeting
2. Mating and phenotyping
3. Discovery of biological function

Reverse genetics dominate

Question 3

What are ES cells?

Answer 3

Embryonic stem cells, sometimes used to induce a knockout

Question 4

How can a laser beam be used to make a knockout? List steps

Answer 4

1. A laser beam is used to make a hole in the zona pellucida of an eight cell embryo
2. The target embryonic stem (ES) cells are introduced into the embryo resulting in generating mice that contain less than .1% host contamination.

The injected ES carrying either homozygous or heterozygous mutations can be directly used for phenotypic analyses.

Question 5

A properly designed DNA vector should contain?

Answer 5

• Promoter and upstream regulatory sequences (5’ end of DNA), can be inducible to allow activation by drugs or light etc.
• Coding sequence, which contains the genetic information for the mRNA to be formed. START and STOP codon needed.
• Termination/polyA signaling sequencing for poly adenylation of mRNA

Question 6

How are tissue-specific inducible transgene expressions made using the rtTA system?

Answer 6

Tatracycline-controlled transactivator (rtTA) system is used to induce gene expression

The system uses a mutated Tn10 tetracycline-resistance operon of E coli. This operon reverses transcriptional repression from the tetR (tet repressor) by preventing binding of tetR to the operator. This induces TetA (the protein that pumps tetracycline/doxycycline antibiotic out of the cell)

Question 7

What is the TetOff and TetOn technique? What is the advantages and disadvantages of these techniques?

Answer 7

TetOff: The target gene is suppressed by tetracycline and starts when supplying an antibiotic is stopped.

TetOn: When doxycycline is present, rtTA binds to tetO and the ORF of interest (of transgene) is being expressed or repressed in the tissue

The major advantage of this method is that expression of the gene can be switched on and off, the disadvantage of the TetOff (older) method is that the animal has to be kept on antibiotics for a long time

Question 8

What are the Cre/loxP and Flp/FRP systems used for? How do they do this?

Answer 8

To create conditioned knock-out.

DNA fragment to be deleted is flanked by two parallel loxP (bacteriophage site) or FRT (flippase recognition target site) sites, expression of Cre (recombinase) or Flp (flippase recombination enzyme) then leads to the excision of the flanked fragment and DNA strands are re-joined by DNA ligase (gene deleted/knocked out)

• loxP (excision by Cre expression)
• FRT (excision by Flp expression)

Question 9

How can the Cre/loxP system be made inducible? How is this system different from the rtTA system?

Answer 9

• Cre is fused to a mutated nuclear estrogen receptor that is unable to bind endogenous hormones but still binds the estrogen agonist tamoxifen or the synthetic steroid RU486
• Fused Cre and Estrogen receptor makes a compled with heat shock protein (hsp90) and is cytoplasmic and inactive.
• Tamoxifen binding activates estrogen receptor, which disrupts the interaction with hsp90 resulting in migratino of Cre into the nucleus and in Cre-mediated excision of target regions flanked by loxP sites.
• Activation of estrogen-tamoxifen Cre/loxP system is irreversible (unlike rtTA system)

Question 10

How can the Cre/loxP system be used to activate gene expression?

Answer 10

• loxP-flanked stop sequence is inserted between the promoter and the gene of interest, preventing transcription.
• Cre excises the stop sequence, permitting promoter activation of the gene of interest

Question 11

How is the genome modified by zinc-finger nucleases?

Answer 11

• Zinc finger nucleases (ZFNs) are designed restriction enzymes made by connecting a zinc finger DNA binding domains with a DNA-cleavage domain (usually restriction endonuclease Fokl)
• Zinc fingers can be designed to target desired DAN sequences, which enables zinc-finer nucleases to target any locus within a genome
• Nuclease domain makes double strain breaks, which are either correctly repaired by homologous recombination or fixed by non-homologous end joining (NHEJ).
• NHEJ is error-prone and can cause either deletion or insertion in the sequence. As an effect the gene function can be entirely or partly disrupted.
• Correctly repaired DNA will be attacked by zinc finger nucleases again until the sequence being repaired will loose its homology and become unrecognizable for nucleases

Question 12

How can zinc finger nucleases be used to promote insertion of genes into specific location by homologous recombination?

Answer 12

There is a small target locus in the genome,

• The donor DNA contains the gene of interest (GOI), a selection marker (puroR) and sequences homologous to the target locus.
• Upon ZFN-stimulated homologous recombinatino at the target site and the insertion of the GOI, the selection marker can be removed by Cre-mediated excision

Question 13

How can gene targeting allow reliable identification, monitoring and manipulation of neurons?

Answer 13

• Green fluorescent protein (GFP) expression can be activated by tamoxifen induced Cre recombination of GABAergic neurons in the cortex.
• Using Cre-inducible light-gated channels enable the photostimulation or photoinhibition of neurons with high spatial and temporal resolution as well as in a cell specific manner

Question 14

What is the brainbow technique?

Answer 14

On the basis of Cre/lox mediated DNA excision or inversion, brainbow transgenes trigger the expression of two to four fluorescent proteins in a cellular population

Question 15

What are DNA transposons?

Answer 15

Pieces of DNA with the ability to change their positions within the genome

Question 16

What are the two general types of transposons?

Answer 16

• Copy and paste transposons (mobilized transcribing an RNA copy, which then becomes reverse transcribed, and is intergrated elsewhere in the genome.)
• Cut and paste transposons (are transposed by the direct excision from DNA and insertion into elsewhere in the genome)

Question 17

How can cut and paste transposons be used as gene vectors?

Answer 17

• Transposons (terminal inverted repeats TIR) can flank the transposase gene
• Bi-component transposon vector system can be used to deliver transgenes that are maintained in plasmids. One component contains DNA of interest between transposon TIRs, carried by a plasmid vector, wherease the other component is a transposase expression plasmid, in which the black arrow represents the promoter driving expression of the transposase
• The transposon carrying a DNA of interest is excised from the donor plasmid and is integrated at a chromosomal site by the transposase

Question 18

What is the application of the sleeping beauty transposon?

Answer 18

A sleeping beauty based gene delivery method for treatment of hemophilia, Fanconi anemia, cancer ischemia and AIDS.

This gene delivery method has a higher success rate than pronuclear injection.

Non random insertion pattern of transposons. There are hotspots and cold regions on a genome wide scale.

Question 19

What are four applications for transposon based gene vectors?

Answer 19

• Cell culture
• Transgenesis
• Insertional mutagenesis
• Gene therapy

Question 20

How can a knockout rat be made using transposons?

Answer 20

• Stem cells are transfected with gene trap transposons and transposase constructs
• Cells with insertions can be selected based on activation of the gene trap marker
• Cells can then be transferred into the testes of sterile males, in which the spermatogonial stem cells with undergo spermatogenesis
• Males are crossed with wild type females to pass the insertinos through the germline and generate knockout animals

Question 21

What kind of carrier has the highest success rate at transfection of a DNA insert? What do you want to prevent with this method?

Answer 21

Transfection using viral carrier

Should prevent recombination-mediated formation of replicative competent retrovirus (RVR) (virises that are able to replicate)

Question 22

How can adult somatic cells be reprogrammed into pluripotent stem cells (iPS)?

Answer 22

By delivery of four transcription factors

• Oct4 and Sox2 (establish pluripotency)
• Klf4 (prevents apoptosis
• c-Myc (immortalize somatic cells and open chromatin)

The inserted genes can be deleted using Cre/loxP system

THe inserted construct that carry the transcription factors to reprogram the cell can be engineered as switchable

Question 23

How can astrocytes by reprogrammed into neuroblasts?

Answer 23

Transcription facts of the SOX family are expressed in neurons from the onset of embryonic development.

Sox2 delivery can reprogram astrocytes into neuroblasts

Question 24

What classes of iRNA can induce gene silencing?

Answer 24

• siRNA
• miRNA
• piRNA (piwi interacting RNA)

Question 25

How does siRNA induce gene silencing?

Answer 25

• dsRNA is cleaved into double stranded siRNAs by dicer

- Target regulation is mediated by the RNA-induced silencing complex (RISC)

Question 26

How does miRNA induce gene silencing?

Answer 26

Several miRNAs are present as clusters in the genome and likely derive from a common pri-miRNA transcript. miRNAs not only mediate mRNA cleavage, but also regulate a variety of biological processes, from housekeeping to response to environment stress

Question 27

How do piRNA induce gene silencing?

Answer 27

They bind Piwi proteins and do not require dicer for their production. piRNA are involved in germ-line development.

Question 28

What is the Argonaute family of proteins?

Answer 28

Ago proteins can bind with siRNAs or miRNAs

When binding with complementary mRNA, the Ago-siRNA or Ago-miRNA complex induces its cleavage and degradation. Imperfect binding induces translational inhibition or deadenylation and subsequent decapping and degradation of the target mRNA.

Question 29

How does phosphorothioate linked DNA (S-DNA) knockdown genes?

Answer 29

Oxygen on each of the phosphate subunits are replaced by sulfur. S-DNA are cleaved by RNase H and off-target effects are commonly observed

Question 30

How do morpholino nucleic acids sterically block and knockdown genes?

Answer 30

5 membered ribose ring has been replaced by 6 member morpholine ring, not degraded in biological systems and has low potential for off-target effect as well as low interaction with the immune system

Morpholino oligos can knock down gene expression, modify RNA splicing or inhibit miRNA activity and maturation

Question 31

What is shRNA?

Answer 31

Short hairpin RNA (vector based and chemically synthesized)

Acts like siRNA with RISC

Question 32

What are two applications for gene silencing?

Answer 32

• Convenient animal model for gene knock-down

- Trials for treatment of cancer, viral infections, AMD and neurodegenerative disease

Question 33

What are two disadvantages of induced gene silencing?

Answer 33

• Off-target effects, where a gene with a similar sequence to target is also repressed
• Overexpression of shRNA can competitively inhibit intracellulara processing of miRNA, which could cause toxic side effects

Question 34

What are zinc finger nucleases?

Answer 34

Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms.

Question 35

How can zinc finger nucleases be used to edit alleles and add DNA constructs?

Answer 35

ZFNs are also used to rewrite the sequence of an allele by invoking the homologous recombination (HR) machinery to repair the double strand break using the supplied DNA fragment as a template.

Question 36

What areas of the brain will respond to the Brainbow technique (don’t need to memorize, just get the point)? What does Brainbow reveal about the brain?

Answer 36

• Purkinje neurons of the cerebellum
• Dentate gyrus of the hippocampus
• Brain stem
• Cortex (layers III to IV)
• Hippocampus CA1

Used to study connectivity between areas of the brain

Question 37

What is another name for transposable elements (transposons)?

Answer 37

Jumping genes

Generally non-coding, but play an important role in development.

Question 38

What is insertional mutagenesis?

Answer 38

Insertional mutagenesis is when transposons function as vectors to help remove and integrate genetic sequences. Given their relatively simple design and inherent ability to move DNA sequences, transposons are highly compatible at transducing genetic material, making them ideal genetic tools.

Question 39

When will there not be expression of a transposon in the genome?

Answer 39

When they are inserted into introns

Question 40

Broadly, how can a sleeping beauty transposon knock-out a gene in a cell?

Answer 40

By inserting a transposon into the ORF of a gene

Question 41

What is a limit of using viral vectors carriers?

Answer 41

• If they can begin to replicate, this can cause issues

- If viral RNA and/or proteins begin to be synthesized, this can cause inflammation and other issues.

Question 42

Give two examples where transcriptional factors can be used to reprogram cells

Answer 42

• Delivering transcription factors to adult stells to produce Induced pluripotent stem cells (iPS cells)
• Inserting Sox2 into astrocytes to reprogram them into neuroblasts

Question 43

How might induced neuroblasts (astrocytes reprogrammed from Sox2) be used in medicine?

Answer 43

Glial cells are most abundant non-neuronal cells in the brain. New proliferative neuroblasts may replace damaged mature neurons in vivo from neural injury or neurodegenerative disease.