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Mouse > Genetic Manipulation > Flashcards

Genetic Manipulation Flashcards

1
Q

How are mutant mice created? (3)

A

Radiation, chemical, transgenesis

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2
Q

What chemicals cause point mutations in DNA? (2) Large lesions that are often multi locus?

A

EMS or ENU. Chlorambucil (CHL).

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3
Q

Define a transgenic mouse. What does transgenic typically refer to?

A

A transgenic mouse is any mouse with foreign DNA integrated into the genome. Typically, a transgenic mouse is one altered through microinjection of foreign DNA into the pronucleus of the egg.

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4
Q

Following implantation of an embryo into a recipient mouse, are all subsequent founder mice genetically identical for the transgene of interest? Why?

A

No. Mice are genetically different as the transgene integrates at random sites, leading to hemizyogus mice. The copy number of the transgene affects the phenotype of each founder and transgene may be lost in subsequent generations.

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5
Q

What is present upstream of a transgene? What agents provide regulatory control?

A

Promoter. Drug-dependent regulatory control with tetra- or doxycycline.

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6
Q

What cells are used for less efficient integration of genetic material? By what method do they integrate?

A

Embryonic stem cells. Homologous DNA recombination

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7
Q

Describe the characteristics of gene trapping.

A

High throughput randomly inserted mutations.

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8
Q

What is the structure of a gene trapping cassette?

A

Vector with gene trapping cassette contains promoter-less reporter gene or genetic marker flanked by upstream 3’ splice site and downstream termination sequence

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9
Q

How does a gene trapping cassette function?

A

Inserted into an intron and is transcribed from an endogenous reporter. The gene is inactivated (termination sequence) and expression of the trapped gene is reported (reporter gene or genetic marker).

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10
Q

How do targeted gene mutations function? What is their function? How is integration monitored?

A

Homologous sequences flank upstream and downstream regions of targeted gene, the construct between may knock out or in the gene. Typically contains reporter gene to track integration.

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11
Q

Where do the agents for site-specific recombination originate from?

A

Cre from coliphage P1 and FLP from Sacchoromyces cerevisiae

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12
Q

How does the Cre/loxP system work? This is an example of what type of mutation?

A

Cre and loxP flank the target gene, with the orientation of the flanking of the loxP sites determining the outcome. Conditional mutation.

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13
Q

What are the steps to create a Cre/lox mouse?

A

Floxed mutation created in ES cells to create a mouse with a conditional mutation. This mouse is crossed with a Cre transgenic mouse. This results in the insertion of reporter genes and selectable markers under the control of inducible gene expression systems.

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14
Q

What can ES cells develop into? From which line were they derived?

A

Pluripotent - any tissue. Derived from 129 mouse known for teratomas.

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15
Q

Where are ES cells injected? What does this create?

A

Injected into inner cell mass of a blastocyst to create a chimera.

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16
Q

What is the goal outcome of chimera creation?

A

Male chimeric progeny produce spermatozoa of ES origin. These chimeras can be mated to the background strain to produce F1 progeny, with N10 generations to create congenic mice on desired background.

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17
Q

How can co-isogenic mice be produced with ES cells?

A

Use ES cells from desired strain, as opposed to 129 (default).

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18
Q

Are most ES lines XX or XY? Why?

A

XY, to favor 129 male chimarism.

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19
Q

What is an aggregation chimera?

A

ES cells are allowed to aggregate with the developing embryo to form the blastocyst in culture, then the chimeric blastocyst is implanted.

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20
Q

What is RNA interference? Why does this system exist?

A

Small interfering RNA (siRNA) finds homologous RNA and interferes. Developed as a natural self-defense for bacteria and archaea against viruses.

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20
Q

What construct functions in induced RNA interference? How is it introduced? What is its function?

A

Small hairpin RNA (shRNA) is introduced into ES cells via electroporation or lentiviral infection. Gene knock-out.

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21
Q

What is the advantage (1) and disadvantages (3) of shRNA interference?

A
  1. Mice are genetically stable but
  2. Transgenesis is never complete
  3. Variable tissue expression
  4. Cannot induce point mutations
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22
Q

What class of molecules are ZFNs and TALENs? What is their general method of action? What do they stand for?

A

Engineering proteins that are fused to nonspecific endonculease Fak1 and target DNA.
ZFN = Zinc finger nucleases
TALENs = Transcription activator-like effector nucleases

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23
Q

Describe the structure of ZFN and how to targets DNA.

A

Consists of 3-6 tandem zinc finger proteins, each of which targets to a specific 3 bp nucleotide sequence. The paired ZFN target opposite DNA strands, allowing dimerization of fok1 and double strand DNA breaks.

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24
Q

What does CRISPER stand for?

A

Clustered regularly interspaced short palindromic repeats

25
Q

Describe the structure of TALENS.

A

Similar to ZFN, with tandem repeats of 33-35 amino acids, each with nucleotide specificity occurring in two hyper-variable amino acids (the repeat variable di-residue) at positions 12 and 13.

26
Q

Describe the CRISPER/Cas system. What does binding cause and how is this damage repaired?

A

RNA-guided endonucleases that target specific DNA sequences and cause double-stranded DNA breaks which are repaired by nonhomologous end joining or homologous recombination. Cas does cutting.

27
Q

Describe nonhomologous end joining.

A

Error prone repair system that results in insertions or deletions with a relatively high frequency, causes gene disruptions

28
Q

Describe homologous recombination. What can introduction of donor DNA lead to?

A

Less common repair mechanism, but manipulation of engineered nucleases can increase incidence. DNA introduction via knock-ins, specific point mutations, or generation of larger modifications.

29
Q

Vectors encoding engineered endonucleases can be introduced via what methods? What are the advantages (3) of this method?

A

Introduction via pronuclear injection of DNA, intracytoplasmic injection of RNA, or transfection of mouse ES cells. Advantages: Creation in any mouse strain without the need to backcross, multiple genes targeted with CRISPER simultaneously, bi-allelic mutations creating functional KO animals in single generation.

30
Q

Transgenes can be broken down into what two categories?

A

Those that are produced via homologous recombination as targeted events at a particular loci and those that occur by random insertion into the genome.

31
Q

What is the difference in ends-in and ends-out gene targeting?

A

Ends-in results in two functional copied of the target gene with the marker gene between. End-out results in two partial sections of the target gene (nonfunctional) with the marker gene between.

32
Q

What forms of radiation are used to create mutants? What are the disadvantages of using this method?

A

Xrays, gamma rays, and neutrons. Low rate of mutation and difficult to use on a large scale.

33
Q

To create a transgenic mouse, pronuclear injection is performed on what pronucleus of a fertilized egg? Usually on what background? What is the result?

A

Male pronucleus. Often FVB (big pronucleus) or C57Bl/6. Results in hemizygote.

34
Q

How are targeted mutations performed?

A

Knock outs occur via DNA sequence insertion into embryonic stem cell (129 or C57Bl/6). DNA taken up via homologous recombination. Stem cell injection into blastocyst, resulting in chimeras.

35
Q

How are ES cells with appropriate homologous recombination and integration selected for?

A

Positive - Allows survival in environment with DNA is integrated and incorporated. Abx (neomycin) resistance
Negative - Present in cells that have integrated DNA, but not incorporated by HR. Sensitivity to thymidine kinase (tk)

36
Q

Describe a gene trap.

A

The insertion sequence activates on insertion, providing a DNA tag for the disrupted locus. Transferring the insertion to the mouse germline to examine function in vivo.

37
Q

What two agents can be used to regulate gene expression?

A

Tetracycline and tamoxifen

38
Q

Describe the Tet operon and its components.

A

Used for temporal and spatial control of transgene expression. Either tTA (tet-controlled transactivator protein) or rtTA (reverse tet-controlled transactivator). Proteins from Tc resistance operon of E. coli transposon Tn10 fused to strong transactivating domain of VP16 from Herpes simplex.

39
Q

Describe the Tet-Off expression system.

A

Uses rTA. Doxy blocks DNA binding, no transcription. Binding results in functioning, no binding due to shape change induced by doxy.

40
Q

Describe the Tet-On expression system.

A

Uses rtTA (altered). Binding prevents transcription. Doxy activates DNA binding and results in transcription.

41
Q

What is a bitransgenic system? How is the tet operon utilized?

A

Conditional gene expression. Put rtTa under tissue specific promoter in mouse 1. Put tet responsive promoter for oncogene expression in mouse 2. Breed together, and get oncogene expression only in specific tissue.

42
Q

How does the Cre-Lox system work in breeding?

A

Cre recombinase will remove select marker genes (loxP). Flank gene of interest with loxP in one mutant. In the second mutant place Cre under a tissue specific promoter or tet operon control. Breed the mutant - the floxed genes of mutant 1 are excised by Cre in specific tissues.

43
Q

Describe the Tamoxifen-Cre system.

A

Without tamoxifen, Cre-ER (mutant ligand binding receptor domain) binds HSP90. When tamoxifen is injected, it displaces HSP90 and binds Cre-ER, resulting in nuclear translocation of Cre-Er and inactivation of target gene.

44
Q

Describe the Flp/Frt system.

A

Similar to Cre/lox system. Flp is recombinase that removes DNA placed between two FRT sites.

45
Q

What does Cre and loxP stand for?

A

Cre = locus that Causes REcombination
LoxP = locus of X over P1. Site on bacteriophage P1 consisting of 34 base pairs.

46
Q

Why would a Cre-LoxP system be used instead of a KO of the gene?

A

Circumvent embryonic lethality caused by systemic inactivation of genes. Experimental control in transgenic animal models to link genotypes to phenotypes.

47
Q

What is a floxed gene?

A

One FLanked on either end by the LOXp sequence.

48
Q

How exactly does Cre recombinase proteins bind?

A

Bind the the first and last 13 base pairs of a lox site forming a dimer. This dimer then binds to a dimer on another lox site to form a tetramer. Lox sites are directional and the two sites joined by the tetramer are parallel in orientation.

49
Q

What occurs when double-stranded DNA is formed by binding of Cre to loxP sites?

A

DNA is cut at both loxP sites by the Cre protein. Strands are rejoined with DNA ligase.

50
Q

For two lox sites on the same chromosome arm, what results in an inversion or deletion of the DNA?

A

Inverted loxP sites result in inversion of the intervening DNA.
A direct repeat of loxP sites results in a deletion event.

51
Q

How can the Cre-LoxP system be used to knock out genes in specific tissues?

A

Requires a mouse strain in which Cre expression is driven by a tissue-specific promoter.

52
Q

What are two characteristics of the CRISPR region of DNA?

A

Nucleotide repeats and spacers. Repeated sequences of nucleotides distributed throughout a CRISPR region.

53
Q

Describe the use of CRISPR by bacteria.

A

In bacteria, the spacers in CRISPR were taken from viruses that previously attacked the organism. They serve as a bank of memories to enable the bacteria to recognize the virus and fight future attacks. Once a spacer is incorporated and the virus attacks, a portion of the CRISPR is transcribed and processed into crRNA.

54
Q

What does the nucleotide sequence of the CRISPR act as?

A

A template to produce a complementary sequence of ss RNA. Each crRNA consists of nucleotide repeat and spacer portion.

55
Q

What is Cas9? What does it bind? What is the function of binding to these sites?

A

Cas9 protein is an enzyme that cutes foreign DNA. Typically binds to two RNA molecules - crRNA and tracrRNA (trans-activating crRNA). Two bound molecules of RNA then guide Cas9 to target site where it will make its cut.

56
Q

How is the section of DNA that will be cut by CRISPR decided?

A

It is complementary to a 20-nucleotide stretch of the crRNA.

57
Q

What type of breaks does CRISPR Cas9 make?

A

Double-stranded breaks, using two separate regions, or domains, on its structure.

58
Q

How are the breaks induced by Cas9 fixed? (2)

A
  1. Non-homologous end joining. Two ends are glued back together. Tends to introduce errors via nucleotide insertion or deletion, resulting in mutations
  2. Filling in gap with a sequence of nucleotide. Cell uses short strand of DNA as a template. Scientists can supply this template, thereby writing-in any gene they want or correcting a mutation.
59
Q

How are specific nucleotide sequences targeted by CRISPR Cas9?

A

Designing the nucleotide sequence of crRNA, which binds to a complementary DNA target.

60
Q

What is a guideRNA in terms of the CRISPR Cas9 system?

A

Fusion of the crRNA and tracrRNA.

61
Q

In the modern CRISPR Cas9 gene editing system, what components are needed? (3)

A
  1. Guide RNA (which contains matching gene sequence
  2. Cas9 protein
  3. Donor DNA (if wanted)

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