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TNS > Exam 3 > Flashcards

Exam 3 Flashcards

1
Q

Forward Genetic Screen

A

Used to identify animals with abnormal phenotypes

Typically done in flies or worms

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2
Q

Reverse genetic screens

A

Takes a known gene and breaks it to observe the outcome

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3
Q

Microarray

A

Used to measure and compare gene expression from biological samples

Extracts RNA from the sample and used an enzyme (reverse transcriptase) to make cDNA

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4
Q

What is cloning?

A

Technique that’s used to make copies of a specific DNA fragment

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5
Q

How does cloning work?

A

It needs the DNA fragment to be isolated using restriction digests or PCR.

Then the gene is inserted into a plasmid and the fragment is inserted into the host cell (bacteria most common)

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6
Q

What is a DNA vector?

A

It is a carrying vehicle that holds an isolated DNA sequence of interest.

Features: can replicate autonomously in host cell and can take on sequences of DNA

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7
Q

What are common DNA vectors?

A

Plasmids or Phages

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8
Q

What is a Plasmid?

A

It is a circle of DNA that contains specific restriction sites, which allow for cutting then insertion. It also indicates if the gene has been inserted.

The restriction site is usually in the middle of a marker

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9
Q

Three main features of vectors?

A

1) An origin of replication
2) Restriction Site
3) Selectable marker

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10
Q

Restriction Enzymes

A
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11
Q

What is Gel Electrophoresis?

A

It is used to isolate, identify, and characterize properties of DNA.

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12
Q

What is PCR?

A

PCR targets and selectively amplifies specific sequences in genomes.

Temperature and polymerase is used to cycle through copy, separate, anneal, and then copy

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13
Q

What are Primers used for in PCR?

A

They are used to target the specific sequence

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14
Q

Steps to PCR?

A

1) Denaturation
2) Annealing
3) Extension: new nucleotides are added to the 3’ end of the extending strand

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15
Q

DNA sequencing

A
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16
Q

What is Southern Blot?

A

It is used to identify presence of known DNA sequence in samples

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17
Q

What is Northern Blot?

A

It’s used to identify presence of RNA in samples

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18
Q

What is Gene Delivery?

A

It is introducing a gene into an organism to observe its function or take advantage of its function

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19
Q

Gene Delivery Strategies

A
  • Physical gene delivery
  • Chemical gene delivery: uses chemicals to porate cells and deliver DNA
  • Viral gene delivery
20
Q

Physical gene delivery techniques

A
  • Microinjection: injecting DNA into the cell with a miniscule probe
  • Electroporation: using an applied electrical field
  • In utero elecroporation: controls gene expression at specific points in development
  • Gene Gun: Gun is aimed at specimen, Gas breaks the rupture disk which drives the bullet down the shaft, Bullet is blocked by the filter screen and DNA-coated metal particles emerge
21
Q

Chemical Gene Delivery

A
  • Calcium Phosphate transfection: Solution with DNA and calcium chloride, HEPES buffered saline solution causes DNA to precipitate out of solution, the solution with precipitate is added to cells
  • Lipofection: lipids formed into liposome around DNA and then caused to fuse with the cell and introduce DNA
22
Q

Viral gene Delivery

A
  • starts with transfecting cells with plasmid encoding viral particles and the gene of interest, after two days, the viral-containing supernatant is collected and a centrifuge is used to collect the viral pellet. then the pellet is resuspended in buffer.
  • Cultured cell line: used to produce copies of plasmid and viral particles
23
Q

What is LacZ and its technique?

A

LacZ is a restriction site.

24
Q

What are reporter genes?

A

They indicate the location of a protein, cell type, or circuit

25
Q

Making transgenic mice

A

It begins with injecting DNA into the recently fertilized egg and the construct is injected into one of the pronuclei. Once the construct is in the egg’s nucleus, it can be injected into a pseudopregnant mouse.

26
Q

What are genes that cause cell death?

A

They are used to kill cells in the brain and can target specific populations of cells

Types:
- Toxins: kills all expressing neurons
(Ex: Ataxin)

Toxin Receptors: when the toxin is applied to the brain, only cells expressing the receptor will die
(Ex: Diphtheria toxin receptor)

27
Q

Genes that are indicators of neural activity?

A
  • Calcium indicators: sensors that change in fluorescence in response to different concentrations of Ca ions
  • Voltage Sensors: sensors that change in fluorescence in response to changes in membrane potential
  • Synaptic activity sensors: sensors that change in fluorescence in response to changes in synaptic vesicle fusion
28
Q

Genes for inducible activation?

A
  • Optogenetics: Membrane-bound channels that depolarie cells when stimulated by light
    (ex: Channelrhodopsin-2 (ChR2))
  • Ligand receptors: Ligand-gated ion channels that depolarize cells whne a ligand is added
    (ex: Capsaicin receptor (TRPV1))

Channelrhodopsin: ion channel that opens with blue light

29
Q

What are knock outs?

A

When gene targeting is used to replace a functional gene with a non-functional sequence

30
Q

RNA interference

A

dsRNA comes into contact with Dicer. Dicer cleaves dsRNA into siRNA. Dicer/siRNA attracts other proteins to form the RISC complex. RISC targets mRNA with a complementary sequence to siRNA and RISC degrades mRNA.

Dicer cleaves RNA and interacts with the RISC complex. RISC interacts with mRNA that’s complementary to the piece it holds.

31
Q

Three ways to target RNAi?

A

1) Deliver dsRNA that is homologous to the mRNA transcript
2) Deliver synthetic siRNAs
3) Morpholinos and siRNAs can also block association and function of mRNA (ribosome blocking production of the protein)

32
Q

What are monoclonal antibodies?

A

They are antibodies that recognize one epitope of an antigen

33
Q

What are polyclonal antibodies?

A

They are antibodies that recognize multiple epitopes of an antigen.

34
Q

How to produce a Monoclonal antibody?

A

The antigen is injected into mice and immunogenic B cells are extracted from the spleen. Immunogenic B cells and Culture myeloma tumor cells are fused to produce hybridomas. Hybridomas are cultured and cells that express antibodies are selected. Those cells are cloned and expand the colony of antibody-producing cells to mass-produce antibodies.

35
Q

How to purify antibodies?

A

The animal is injected with antigen and time is allowed for B cells to produce antibodies. Then blood is removed from the animal and put in a centrifuge to collect blood serum. The antibodies are purified on a column that contains the antigen.

36
Q

What is Western Blotting?

A

It is when an antibody binds to a protein that’s been run on a polyacrylamide gel to mark the presence, approximate size, and relative quantity of the protein in a sample.

37
Q

Enzyme-linked immunosorbent assay (ELISA)

A

Antibody binds to a protein to mark the presence and measure the quantity of the protein in a sample

Antibody binds to bottom surface of a plate. Sample is added to the plate and any protein that binds to the antibody, is adhered to the surface. Sample is washed away but protein remains. A second antibody linked to an enzyme, is added and converts the chemical substrate into chromogenic or fluorescent signal. Then optimal device is used to measure the signal and determine quantity of protein present.

38
Q

Radioimmunoassay (RIAA)

A

The antibody binds to radioactive proteins of known concentration. A sample that contains nonradioactive proteins is added to compete with the radioactive proteins, which allows for determining the protein concentration in a sample.

39
Q

Immunohistochemistry

A

Antibody binds to a protein to show spatial expression in tissues or cells

40
Q

Immunoprecipitation

A

An antibody is bound to small beads. A sample is mixed with the beads causing the anti-body to bind a purify a protein. The protein sample is then eluded off the beads.

41
Q

Coimmunoprecipitation

A

An antibody is bound to small beads. A sample is mixed with the beads causing the antibody to bind and purify a protein. After the protein is eluded, a separate antibody in conjunction with a Western blot or ELISA is used to detect the presence of any secondary proteins that could interact with the immunoprecipitated protein.

42
Q

ChIP (Chromatin immunoprecipitation)

A

Demonstrates if a protein interacts with specific regions of DNA

An antibody is bound to small beads in a column and a sample is run through the column. This causes the antibody to bind and purifies a protein. After the protein is eluded, PCR is used to determine the presence of any bound DNA sequences.

43
Q

Chromatography

A

It is when a sample is run though a column that’s composed of porous membrane or gel.

Different columns = different properties

Allows proteins to be separated

44
Q

Types of Chromatography

A

Gel-filtration Chromatography: proteins are separated based on size

Ion-exchange Chromatography: proteins are separated based on their charge

Affinity Chromatography: uses small molecules to bind to proteins that may have high affinity

45
Q

Steps for genetic knockout?

A

1) Creat targeting construct
2) Inject construct into ES cels and select the cells that incorporated the construct
3) Insert ES cells into blastocyst
4) Insert blastocyst into female mouse
5) Breed offspring until successful knockout achieved

46
Q

JAK-STAT pathway

A

Cytosine binds to its receptor, JAKs transfers a phosphate to other proteins and the phosphate group causes a conformational change in STATs. STATs bind to the receptor and JAKs add another phosphate group to the STAT proteins. This causes them to dissociate from the receptor and bind to each other. The STAT diners teams locate to the nucleus and activate target genes.

TNS (3 decks)

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