Gene technology

Question 1

Based on what are DNA fragments separated on gel electroforeses?

Answer 1

Based on size

Question 2

What does ethidium bromide do?

Answer 2

It visualizes DNA fragments

Question 3

What would a large fragment stained with ethidium bromide look like compared to a smaller fragment?

Answer 3

The larger fragment emits more light

Question 4

What type of RNA is most abundant?

Answer 4

ribosomal RNA rRNA

Question 5

How are proteins detected during a western blot?

Answer 5

Using an antibody

Question 6

Based on what does an SDS-page gel separate proteins?

Answer 6

Based on size

Question 7

What are the 4 steps of southern blotting?

Answer 7

1 run a DNA gel to separate fragments based on size
2 transfer fragments from the gel to a filter (blotting) so that the probe can access the fragments
3 incubate the filter in a solution containing the probe and remove excess unbound probe
4 visualize probe

Question 8

can southern blotting be used to determine if a gene witha known sequence and a gene with an unknown sequence are homologs?

Answer 8

Yes, if the sequence of the suspected homolog is similar enough the probe designed for the known sequence will also bind to this homolog

Question 9

Why is taq polymerase used in PCR?

Answer 9

For PCR DNA fragments have to be separated, this is done at 94 C, taq polymerase is stable at 94 C

Question 10

How many times does double stranded DNA double per PCR cycle? How many fragments would you have after 30 cycles?

Answer 10

Every fragment doubles per cycle, after 30 cycles you would have 2^30 fragments

Question 11

Suppose you know the amino acid sequence of a protein. Is it in principle possible to use PCR to amplify a (partial) DNA sequence of the gene that encodes this protein?

Answer 11

Yes, eventhough most amino acids can be coded by different codons, it is still possible to design specific primers to amplify this sequence with PCR.

Question 12

What are degenerate primers?

Answer 12

A primer mix containing primers with slightly varying nucleotide sequences

Question 13

How is cDNA synthesized

Answer 13

Reverse transcriptase is an enzyme that can use RNA as a template to produce cDNA. Like other DNA polymerases, it needs a primer to start synthesizing the cDNA strand. Therefore, an oligo(dT) primer is added, which binds to the poly(A) tails of the mRNA molecules. After production of the first cDNA strand, the mRNA strand has to be destroyed and replaced with DNA. RNase creates nicks and gaps in the mRNA. DNA polymerase I then uses the remaining double stranded fragments to synthesize the second cDNA strand. On its way it removes remaining mRNA by hydrolyzing it.

Question 14

What primer does reverse transcriptase need, where does it anneal?

Answer 14

Oligo-dt primer, it anneals to the poly-A tail
this is not the only possible primer, but by far the most used one

Question 15

How do you obtain a promoter region for a gene?

Answer 15

PCR amplify genomic DNA
Ligate this DNA into cloning vectors
Transform bacteria with cloning vectors
Multiply in bacteria

Question 16

What is DNA cloning?

Answer 16

Isolating a DNA fragment and fusing it into another DNA sequence of choice

Question 17

What characteristics make vectors suitable for cloning DNA fragments, that are obtained upon digestion with restriction enzymes, in E coli?

Answer 17

• autonomous replication
• unique restriction sites
• genes that confer antibiotic resistance
• small size

Question 18

Can ethidium bromide be used to visualize RNA fragments?

Answer 18

Yes, if the gel was not run under denaturing conditions. Under non-denaturing (normal) conditions, the single stranded RNA molecules form secondary structures by internal base pairing. Ethidium bromide can bind to these double stranded parts.

Question 19

What techniques are used to check whether you have obtained the correct DNA recombinant and why?

Answer 19

• DNA sequencing -> With a specific primer a DNA sequence of the recombinant DNA construct can be obtained
• PCR -> with PCR it is possible to check whether the products that are obtained with specific primers have the predicted size(s)
• DNA digestions -> with specific restriction enzymes it is possible to check whether the products that are obtained after DNA digestion have the predicted size(s)

Question 20

Suppose you want to detect a specific human protein (SHP) on a Western blot on which a human protein sample was loaded. You have an unlabeled antibody against this human protein that was made in rabbit. Which secondary antibody would you use for the detection?

Answer 20

Labeled mouse-anti-rabbit antibodies

Question 21

When can you determine the expression of a gene using a northern blot?

Answer 21

When the gene sequence of the gene is known and a probe can be made.

Question 22

You also you want to obtain large amouts of the same interesting mouse protein, so that you can study binding affinities with other proteins, study its enzymatic activity and produce antibodies against it, etc. Therefore you decide to try to produce the protein in a prokaryotic expression system. Which approach(es) can you use to obtain IMP DNA that can be used in such expression system?

Answer 22

Use PCR on cDNA (RT-PCR) for amplification, ligate the product into a PCR cloning vector and multiply it subsequently in bacteria.

Question 23

When making a transgenic mouse by inserting an extra copy of a gene do you need to select for homologous recombination?

Answer 23

It is not necessary to select for homologous recombination, because the endogenous gene does not have to be changed and it is not necessary to integrate the construct in a specific region of the genome.

Question 24

When making a transgenic mouse by inserting an extra copy of a gene do you need to use embryonic stem cells?

Answer 24

It is not necessary to use embryonic stem cells, because it is not necessary to select for homologous recombination. In this case it is better to inject the construct directly into the oocyte because this method is more straightforward and faster (no step with chimeric mice).

Question 25

How do you determine the expression level of a transgene?

Answer 25

The expression levels can be determined with Reverse Transcription combined with PCR, Northern blot or Western blot analysis and not with Southern blot analysis. If one technique has to be selected, Western blot analysis is slightly preferred, because the final NMDA receptor levels are important and not the levels of its RNA (even though usually these levels correlate reasonably well).

Question 26

Do chimeric mice form when a construct is injected directly into the oocyte?

Answer 26

No, chimeric mice to not form when micro-injection is done directly into the oocyte

Question 27

Do mice survive a western blot if expressions are tested for a brain function?

Answer 27

No

Question 28

Why do you need to cross F0 mice with WT mice in order to continue to experiment?

Answer 28

Crossing mice is necessary to ensure that you can do experiments with several genetically identical mice, and also do additional experiments later on. This is also the reason why you select mice that contain only one insertion of the transgene in their genome.

Question 29

do you need a viral vector when injecting a construct into an oocyte?

Answer 29

no

Question 30

Is it possible to create a transgenic mouse that overexpresses the NMDA receptor in the hippocampus via the transfection of embryonic stem (ES) cells instead of via injection into a fertilized oocyte?

Answer 30

yes

Question 31

if you wish to research that if NMDA overexpression leads to higher learning capabilities, how would the experiment design look?

Answer 31

1. Make a construct; 2. Microinject the construct into fertilized oocytes and grow the mice (F0); 3. Determine the genotype of the F0 mice; 4. Cross transgenic F0 mice with WT (=wild type) mice; 5. Determine the genotype of the offspring (F1); 6. Test the learning capabilities of transgenic F1 mice. 7. Test the expression levels of the inserted gene in transgenic F1 mic

Question 32

Suppose you have a construct that consists of a coding sequence of a gene, that naturally occurs in the mouse genome, coupled to a promoter that is different from the normal promoter, but that also does naturally occur in the mouse genome. Is it possible to use PCR to determine whether this construct is integrated into the genome of a mouse?

Answer 32

Yes, The forward primer should be specific for the promoter, the reverse primer for the coding sequence of the gene.

Question 33

What 3 different techniques can be used to test for gene expression and why?

Answer 33

- Western blot analysis, Western blot analysis can be used to study protein levels. - Northern blot analysis, Northern blot analysis can be used to determine RNA levels, and usually these levels correlate reasonably well with protein levels. - RT-PCR (Reverse Transcription PCR) Reverse transcription coupled to PCR (RT-PCR) can be used to determine RNA levels, and usually these levels correlate reasonably well with protein levels.

Question 34

When making a knock out, is it necessary to select for homologous recombination?

Answer 34

Yes, to make a knock out you want to replace the endogenous gene with something else, therefore you must select for homologous recombination

Question 35

What does the experimental scheme for creating knock out mice with t-bet?

Answer 35

- Make a targeting construct - Electroporate construct into ES cells - Grow ES cells in selective medium/determine genotype - Inject ES cells into blastocyst/grow F0 mice - Cross chimeric F0 mice with WT - Determine the genotype of F1 mice - Intercross heterzygous F1 mice - Select and intercross knockout mice - Further analyze knockout mice

Question 36

What are the elements you want to use for making a targetting construct that can be used to create a KO mouse?

Answer 36

Thymidine kinase gene (TK) (negative selection marker, only stays in the gene with random insertion) neomycin resistance gene (neo) (positive selection marker) 5'-end and 3'-end of the T-bet gene

Question 37

What are the possible ways of inserting DNA into ES cells?

Answer 37

electroporation, lipofection or transfection

Question 38

What is the two stage procedure for Production of gene-targeted knockout mice?

Answer 38

1. In the first stage, a DNA construct (also called targeting vector) containing a disrupted allele of a particular gene X, a neomycine resistance marker and a tk marker (see Fig. 3-1) is introduced into ES cells. Recombinant cells are selected by treatment with G418, since cells that fail to pick up DNA or integrate it into their genome are sensitive to this cytotoxic compound. The surviving cells are then subjected to treatment with gancyclovir. Only ES cells that undergo homologous recombination can survive in the presence of both G418 and gancyclovir (see Fig. 3-1). 2. In the second stage in the production of knockout mice, ES cellsheterozygous for a knockout mutation in gene X are then injected into a recipient wild-type mouse blastocyst that subsequently is transferred into a surrogate pseudopregnant female mouse. The resulting progeny will be chimeras, containing tissues derived from both the transplanted ES cells and the host cells (see Fig. 3-3). If the ES cells are also homozygous for a visible marker trait (coat colour) then chimeric progeny in which the ES cells survived and proliferated can be easily identified. Chimeric mice are then mated with mice homozygous for another allele of the marker trait to determine if the knockout mutation is incorporated into the germ line. Finally, mating of mice, each heterozygous for the knockout allele, will produce progeny homozygous for the knockout (disrupted gene) mutation.

Question 39

How can tissue/cell specific knock outs be done?

Answer 39

The loxP-Cre recombination system can knock out genes in specific cell types. Two loxP sites are inserted on each side of an essential exon (2) of the target gene X by homologous recombination, producing a loxP mouse. Since the loxP sites are in introns, they do not disrupt the function of X. The Cre mouse carries one gene X knockout allele and an introduced cre gene from bacteriophage P1 linked to a cell-type-specific promoter. The cre gene is incorporated into the mouse genome by nonhomologous recombination and does not effect the function of other genes. In the loxP-Cre mice that result from crossing, Cre protein is produced only in these cells in which the promoter is active. Thus these are the only cells in which recombination between the loxP sites catalyzed by Cre occurs, leading to deletion of exon 2. Since the other allele is a constitutive gene X knockout, deletion between the loxP sites results in complete loss of function of gene X in all cells expressing Cre. By using different promoters, researchers can study the effects of knocking out gene X in various types of cells.

Question 40

What is a knockin mouse?

Answer 40

A mouse that does not have a knockout gene, but a knockin gene which is transcribed the same as the wild-type, but the protein that it encodes has a different function.

Question 41

Where does the loXP-cre system come from?

Answer 41

It comes from bacteriophages, but also functions in mice

Question 42

What is the loxP-cre system used for?

Answer 42

To research cell/tissue specific knockouts that would be lethal using conventional knock-outs

Question 43

What is luciferase?

Answer 43

The luciferase gene from the firefly Photinus pyralis (LUC) encodes a 62 kD protein. In a two-step reaction, it combines ATP with the substrate luciferin to yield an enzyme-bound intermediate complex, which then undergoes oxidative decarboxylation to produce the highly luminescent compound oxyluciferin (OL), which is capable of releasing a photon at 562 nm It is the enzyme involved in bioluminescence

Question 44

What is the difference between LUC, RLUC and LUX?

Answer 44

LUX from bacteria and RLUC from the coral Renilla are unrelated to firefly LUC and importantly require different substrates

Question 45

What is a reporter gene?

Answer 45

genes with easily detectable protein products used for detecting a particular gene activity

Question 46

How are reporter genes used?

Answer 46

The promoter of the gene of interest is spliced (using restriction-enzyme-based “cutting and pasting” of recombinant DNA molecules) onto the coding region of the gene of interest. After the gene fusion construct to the gene of interest the reporter gene will announce the activity of the gene of interest in the appropriate tissue and when it is active as defined by its promoter

Question 47

What is GUS?

Answer 47

b-glucoronidase, coded for by the gene UidA in E. coli. A popular reporter gene for plants.

Question 48

What are the 2 methods of using GUS as a reporter gene?

Answer 48

1 By using a fluorogenic substrate such as MUG -> this is cleaved by GUS yielding MU which is fluorescent 2 By using histo-chemical substrate X-gluc. This is used for cellular localisation of GUS gene expression. This only works if no absolute quantification of GUS is needed. It turns blue upon hydrolising and can easily be fixated unto tissue which makes it good for identifying promoter expression patterns

Question 49

How is X-gluc visualised?

Answer 49

UidA catalyses the cleavage of this substrate, generating the colorless and soluble product 5-bromo-4-chloroindole. This, in turn, is readily oxidized and dimerized to form insoluble indigo, which is dark blue in colour. A great advantage of this substrate is that the indigo is easily visible by eye, needing no specialized equipment, although close obser- vation of tissues requires at least a low-power light microscope.

Question 50

How is T-DNA transferred into wounded plant cells?

Answer 50

When a plant cell is wounded, it releases factors that stimulate transcription of the vir genes on the Ti plasmid that function in the transfer of T-DNA into the plant cell. Only the T-DNA region of the Ti plasmid is transferred to the plant cell. T-DNA is bounded by 25-bp imperfect repeats termed the left border (LB) and the right border (RB). Transfer begins with a nick in the DNA strand in the RB, then a nick occurs at the LB producing a single-stranded T-DNA molecule. The T-DNA molecule enters the plant cell, where it integrates randomly into the chromosomal DNA. The single-stranded T-DNA region of the Ti plasmid is repaired by DNA replication, so the Agrobacterium has not lost any information by transferring DNA to the plant cell.

Question 51

What is a polylinker?

Answer 51

A polylinker is a chemically synthesized DNA segment that contains a series of unique restriction sites that can be used to produce sticky ends for inserting the DNA segments to be cloned.

Question 52

What is bombardment?

Answer 52

Direct transfer of DNA into plant cells by shooting a very tiny metal bullet coated in DNA into the plant. The directly hit plant cells will die, but the surrounding cells can take up the DNA

Question 53

What are the steps for A. tumefaciens-mediated transformation of plant cells ?

Answer 53

1 insertion of the gene of interest into the cloning site of the binary vector using basic in vitro DNA recombinant techniques. 2 transfer of the recombinant plasmid into E. coli and selection of transformants. 3 transfer of the plasmid out of E. coli into recipient A. tumefaciens strain carrying a modified (defective, or disarmed) Ti-plasmid that contains a complete set of vir genes but lacks portions or all of the T-DNA region, so that this T-DNA cannot be transferred. Transformation of such a recipient A. tumefaciens strain can be accomplished by either mating of A. tumefaciens and E. coli or by electroporation of the naked, recombinant DNA plasmid into A. tumefaciens. 4 transfer of the T-DNA region of the binary vector carrying the gene of interest into (wounded) plant cells 5 selection and regeneration of transformed plant cells

Question 54

Starting at LB and going clockwise, what are the parts of a Ti-plasmid?

Answer 54

LB, aux synthesis genes, cytikinin synthesis genes, opine synthesis genes, RB, opine catabolism, ORI, Virulence genes

Question 55

What are LB and RB?

Answer 55

right/left border of the T-DNA region. It flanks the T-DNA and is about 25 bp long. they are involved in exercision of T-DNA sequence

Question 56

What is T-DNA exercision?

Answer 56

a 2 stage process in which RB is nicked between the 3rd and 4th bp repeat, then LB is also nicked leaving a single stranded T-DNA which is then released

Question 57

What are reverse genetics?

Answer 57

determining the expected phenotype based on the genotype

Question 58

What are forward genetics?

Answer 58

Determining the genotype based on the phenotype

Question 59

How do you start and maintain a packaging cell line?

Answer 59

Isolate individual cells by disrupting the extracellular matrix and cell-junctions: proteolytic enzymes trypsin & collagenase to digest matrix. EDTA solution to chelate Ca2+ on which cell-cell adhesion depends. Mammalian cells require a solid surface that is coated with material they can adhere to (e.g. polylysine or extracellular matrix components). This property can be used to obtain specific cell types based on surface properties andtheir binding to antibodies

Question 60

What is a Fluorescence-activated cell sorter?

Answer 60

a +, - or neutrally charged GFP attached to a cel sorted in tubes by a magnetic field

Question 61

What do you do with a Laser micro-dissection microscope?

Answer 61

Cut out colored parts of a plate

Question 62

What is a cell line?

Answer 62

an amount of cells that is genetically identical and divides infinitely

Question 63

What is electroporation?

Answer 63

Salt solved in water creates holes in the cell wall

Question 64

What is lipofection?

Answer 64

The construct fuses with lipid membrane

Question 65

What is an adenovirus?

Answer 65

Adenovirus is used as a viral vector by deleting essential viral genes, inserting a gene of interest, packaging the virus in helper cells, and using the resulting particles to efficiently deliver the gene into host cells without integrating into the genome.

Question 66

What is cotransformation?

Answer 66

the use of 2 constructs simultaneously to transform cells

Question 67

What is a packaging cell line?

Answer 67

Cells expressing viral proteins into which viral RNA can packaged into infectious virus particles to express foreign genes tumor cells are well suited due to infinite replication