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CORE Block 3 > Gene Targeting > Flashcards

Gene Targeting Flashcards

1
Q

What is the definition of gene targeting?

A

Production of a specific change directly in the gene of an organim.

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2
Q

Describe KO versus KD in C. elegans.

A

Worms will eat E. coli containing different interfering RNAs. This changes the phenotype of the worm, because the RNA will knock down the expression of the DNA it is complementary to. However, the RNA does not change the gene itself, so it is not a knock out.

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3
Q

Why is genomic DNA rather than cDNA used for gene targeting into a model organsim?

A

Because it is targeted, you want extreme homology, including that to promotors, enhancers, and introns.

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4
Q

What considerations need to be made prior to inserting DNA into ES cells?

A
  1. characteristics of the cell you want to target (how can DNA get in?)
  2. How the cell processes the DNA
  3. how the transfected cells contribute to all the tissues of the developing organism
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5
Q

What transfection method is typically used for ES cells?

A

Electroporation

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6
Q

Describe mouse embryonic stem cells.

A

They are made from the iner cell mass of the embryo, and are pluripotent (cannot form the trophoblast surrounding them). The cells must be returned to the inner cell mass of a mother who will carry them to a full organism.

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7
Q

What are the characteristics of mouse ES cells used for gene targeting?

A
  1. harvested from inner cell mass of blastocyst or purchased
  2. can be maintained in culture and maintain ability to differentiate, even into gametes
  3. are XY because males have greater reproductive capactiy
  4. Derived form mouse with different fur color than mouse in which they will develop to screen for chimera.
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8
Q

How is the targeting vector made for insertion or replacement?

A

-selectable marker inserted between regions of homology with a linearized point outside the region of homology. Higher homology facilitates HR, so ideal targeting vector is constructed from gDNA library from same mouse strain as the ES cell line!

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9
Q

Describe the positive and negative selectable markers for mouse ES cells.

A
  • Positive: neomycin phosphotransferase, hygromycin phosphotransferase, puromycin
  • negative: thymidine kinase, cytosine deaminase (these will be cut out from vector upon successful transfection)
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10
Q

How are negative selectable markers lost in ES cell gene targeting?

A

They are in regions of no homology, so they will not be integrated into the genome of the mouse ES cells.

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11
Q

Why would random integration of a targeting vector containing a negative selectable marker be bad?

A

Because the marker will not be lost upon integration into the genome. Even if the positive marker is still there, the negative marker will not pass the test.

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12
Q

How are randomly inserted vectors in mouse ES cells selected against?

A

All targeting vectors will have positive and negative selectable markers. If the vector is randomly inserted, the negative marker will be expressed and the cells will be susceptible to drugs.

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13
Q

Which selectable markers are used if we want to do NHEJ?

A

Just positive selectable marker.

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14
Q

How can southern blotting be used to test for successful homologous recombination?

A

Oftentimes the endogenous gene sequence will have a different number or type of restriction sites than the targeting vector, so DNA from non transfected and transfected cells can be treated with restriction enzymes and run on a gel and probed with regions of homology outside areas of difference.

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15
Q

When are mouse ES cells collected for the insertion of targeting vectors by electroporation?

A

3.5 days p.c.

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16
Q

Describe the implantation of homologously recombined ES cells.

A
  • blastocysts rest for a few hours
  • injected blastocysts transferred to uterine horn of 2.5 days pc pseudopregnant mouse
  • some transferred blastocysts will develop into healthy pups.
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17
Q

Describe selection of homologously recombined mice after implantation and development.

A

ES cell line was generated from mouse with brown hair and injected into mouse with black hair (recessive). Therefore, you will choose a chimeric male mouse to use as the founder for reproduction. These mice chosen should be mostly brown (dominant trait). Chimera arises due to some ES cells coming from the donor mother and the developmental mother.

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18
Q

What steps must be taken to reproduce chimeric, transgenic mice?

A

The Male embryos are selected for and implanted, and the chimeric males are bred to wildtype females. Their litter will have the potential to be the first generation with all cells to have one allele of the transgene. These progeny must be interpred in order to produce homozygouse mice for the transgene. Western blotting can show if the gene has been knocked out or overexpressed if added.

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19
Q

Which generation of mice may be homozygous for a transgene?

A

F2

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20
Q

How can you overcome a gene KO that is embryonic lethal?

A

You can couple the knockout to a Cre/lox system, in which you induce the removal of a gene at a certain point, such as during adulthood.

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21
Q

Describe Cre recombinase activity.

A

It is a nuclease that recognizes loxp sites flanking a gene of interest. The homologous loxp sites align, and Cre cuts at them, effectively excising the sequence that was between them and leaving just one loxp sequence behind.

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22
Q

How can Cre/lox be used to perform tissue-specific gene inactivation?

A

Make one plasmid with the loxp sites flanking the exon you want to remove (must have high amounts of homology in order to keep it tissue specific). Use whole gDNA of the gene of interest in order to keep promotors and enhancers that contribute to it being gene specific. Will introduce to ES cells. Will screen cells for integration using genotypic screening such as southern with restriction digests or with phenotypic screening such as positive or negative selectable markers. Breed with mouse that has Cre recombinase gene downstream of a tissue-specific promotor.

23
Q

Is the mouse with the Cre plasmid transgenic or gene targeted?

A

It is transgenic. As long as Cre has the promotor of interest, it does not matter where it enter the genome.

24
Q

Describe breeding to get mouse that is homozygous for Cre/lox system.

A

After implantation of the chimeric balstocyst into the mother, screen her pups for chimeric fur color. The males that are most chimeric will be bred with wild type females. These will be mostly heterozygous. Then interbreed these to get homozygotes. Once we establish this cell line for both plasmids, then breed the two cell lines (Cre and Lox)

25
Q

Once a loxp plasmid-containing mouse line is made, how can you study the function of the floxed gene in different tissue types?

A

Just breed the mice with a Cre-transgenic mouse line that has different promotors and enhancers from the tissue you are interested in.

26
Q

How can you determine heterozygote versus homozygote Cre/lox mice from breeding?

A

Can take tail biopsy and do southern to determine whether there is a change in the DNA sequence (using restriction sites).

27
Q

Name the applications of Cre/lox.

A
  1. general knockout (contitutive Cre expression)
  2. inducible knockout (for a certain time)
  3. tissue specific knockout (using tissue-specific promotor)
  4. Removal of selectable marker gene that might affect chromosome stability and affect results of the study.
28
Q

What does TALEN stand for?

A

transcription activator-like effector nuclease (the nuclease part can change to use other functional domains such as activators or repressors)

29
Q

What is the difference between TALEN and ZFN sequence recognition?

A

ZFN modules recognize 3 nucleotide sequences, while each TALEN repeating unit recognizes a single specific nucleotide.

30
Q

What is FOK1?

A

It is the nuclease attached to ZFN and TALEN modules that performs the cutting on either strand, lending to a ds break.

31
Q

What are the repeating units of the ZFN’s?

A

They are alpha helices and beta sheets. The alpha helices recognize the 3-nt sequence that we want.

32
Q

Why might we want to make a ds break using TALEN or ZFN?

A

To allow for homologous recombination with a plasmid vector, or so that it chews itself back within an exon, for example, to knockout a gene.

33
Q

Which part of the TALEN binds DNA?

A

The hypervariable region of the TALE repeat domains.

34
Q

Why might the sequence for TALEN proteins be difficult to clone in bacteria?

A

Because of the high sequence homology between TALE repeat domains. Bacteria do not always like this type of extensive homology.

35
Q

Describe the general targeting strategy with ZFNs or TALENs.

A
  1. Identify the DNA sequences in the gene we will be targeting for the ds break (can probe for exons in the cDNA of the gene)
  2. Generate ZFN or TALEN expression vectors and clone them into bacteria
  3. obtain the mRNAs encoding these plasmids in an in vitro transcription.
  4. Microinject the mRNAs into a fertilized egg.
  5. Screen progeny for targeted/modified allele (should all be homozygous, can do Southern blotting, or even western for a KO since they should be homozygous)
36
Q

Name two large benefits of ZFN and TALENS.

A
  1. No need to test for incorporation of mRNAs into fertilized egg, just check the pups.
  2. all pups should be homozygous for the modified allele, so no extensive breeding is required.
37
Q

Describe what can be done with one pair of TALENs.

A
  • gene disruption and resealing by NHEJ: ressecting the ends of the dsbreak
  • gene replacement by HR: vector with homology to the region being cut
  • gene addition by HR
38
Q

Describe what can be done with two pairs of TALENs.

A

Allows for manipulation of larger segments of the genome

  • Can cut out a region of a chromosome and delete it (ends come together by NHEJ)
  • can cut out a region of the chromosome and invert it (NHEJ)
  • Can cut on different chromosmes and do translocation (NHEJ)
39
Q

How would a TALE with a repressor rather than a nuclease affect the region being broken?

A

We will have a KD rather than a KO.

40
Q

Using TALENs, ZFNs, and CRISPR, when is the injected fertilized egg introduced into the pseudopregnant mother?

A

Introduced to the oviduct at 0.5 days p.c.

41
Q

Describe the overall schematic of CRISPR-Cas9.

A

guide RNA has homology to a sequence of interest, and will guide Cas9 there. Cas9 endonuclease will then cleave the DNA on both strands.

42
Q

Where does CRISPR originate?

A

In bacteria as an innate immune response to foreign DNA such as from conjugation from other bacteria or from transduction by a bacteriophage.

43
Q

What step required for TALEN and ZFN can CRISPR skip?

A

It does not need to be incorporated into a plasmid. We just will make the guide RNA and Cas9 mRNA from the known DNA sequence (using RNA polymerase, NTPs, primers, DNA sequence, and T7 or other bacterial promotor.

44
Q

What is needed for a gene deletion or replacement using CRISPR?

A

Two pairs of CRISPR guide RNAs.

45
Q

How does a gene deletion or replacement using CRISPR recombine?

A

Gene deletion is NHEJ. Replacement is HR.

46
Q

Is gene editing using CRISPR targeted?

A

Yes, especially if you only want to change a single aa, can cut on either side of the codon.

47
Q

Is HR high or low in eukaryotes?

A

It is high in yeast, but very low in mammals.

48
Q

Why do we use ES cells for gene targeting with classical methods?

A

Because they are pluripotent and we can screen for HR easily. This is important especially in mice because the rate of HR is very low.

49
Q

In what model organisms does CRISPR work?

A

planst, fish, worms, drosophila, yeast, bacteria, mammals.

50
Q

How can HIV infections be treated with ZFN?

A

It can be used to cut and knockout the CCR5 gene sequence so the receptor to HIV is not expressed in the cell membrane and HIV cannot enter the cell. This can also be done with CRISPR.

51
Q

How can cancers be treated with gene targeting or modification?

A

CRISPR, to delete or modify drivers of cancer. However, delivery system remains an issue as do potential off-target effects. There are also ethical concerns involved with modification of the human genome.

52
Q

How is DNA obtained form a DNA library?

A

Hybridize a probe of the sequence of interest to identify colonies that have the DNA you want to purify and use.

53
Q

How is cDNA made only from messenger RNA?

A

The other types of RNA do not have a poly-A tail.

CORE Block 3 (5 decks)

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