Genetic Manipulation

Question 1

How are mutant mice created? (3)

Answer 1

Radiation, chemical, transgenesis

Question 2

What chemicals cause point mutations in DNA? (2)

Answer 2

EMS or ENU

Question 3

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

Answer 3

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.

Question 4

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

Answer 4

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 and the transgene may be lost.

Question 5

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

Answer 5

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

Question 6

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

Answer 6

Embryonic stem cells. Homologous DMA recombination

Question 7

Describe the characteristics of gene trapping.

Answer 7

High throughput randomly inserted mutations.

Question 8

What is the structure of a gene trapping cassette?

Answer 8

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

Question 9

How does a gene trapping cassette function?

Answer 9

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).

Question 10

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

Answer 10

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.

Question 11

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

Answer 11

Cre from coliphage P1 and FLP from Sacchoromyces cerevisiae

Question 12

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

Answer 12

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

Question 13

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

Answer 13

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.

Question 14

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

Answer 14

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

Question 15

Where are ES cells injected? What does this create?

Answer 15

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

Question 16

What is the goal outcome of chimera creation?

Answer 16

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.

Question 17

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

Answer 17

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

Question 18

Are most ES lines XX or XY? Why?

Answer 18

XY, to favor 129 male chimarism.

Question 19

What is an aggregation chimera?

Answer 19

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

Question 20

What is RNA interference? Why does this system exist?

Answer 20

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

Question 20

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

Answer 20

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

Question 21

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

Answer 21

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

Question 22

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

Answer 22

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

Question 23

Describe the structure of ZFN and how to targets DNA.

Answer 23

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.

Question 24

What does CRISPER stand for?

Answer 24

Clustered regularly interspaced short palindromic repeats

Question 25

Describe the structure of TALENS.

Answer 25

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.

Question 26

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

Answer 26

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.

Question 27

Describe nonhomologous end joining.

Answer 27

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

Question 28

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

Answer 28

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.

Question 29

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

Answer 29

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.