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Genetic Engineering Flashcards

1
Q

What are the 6 types of gene therapies?

A
  1. production of recombinant therapeutic proteins (most common)
  2. production of genetically engineering Abs (common cancer treatment)
  3. production of genetically engineered vaccines
  4. production of reagents for gene therapies
  5. testing of treatment in genetically modified animals
  6. genetic modification of patient or donor cells in gene & cell therapy
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2
Q

What are 2 ways to add exo-siRNA?

A
  • Just add the siRNA itself (temporary)
  • Have an shRNA expression vector (permanent)
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3
Q

What is transfection?

A

Adding nucleic acids (DNA or RNA) to cell but doesn’t tell you about the fate, or effect

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4
Q

What is transformation?

A
  • The DNA integrated into the genome, changing cell fate (PERMANENT)
  • shRNAi expression vector integrated into genome
  • NO transformation with siRNA treatment
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5
Q

What does shRNAi lead to?

A

suppression of transcription

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6
Q

What is associated with dysplasia?

A

knock out mouse lacking EVC cells

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7
Q

What do gene targeting vectors replace?

A

exons

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8
Q

What is meant by the term “knock in”?

A

Put something in place of the endogenous gene

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9
Q

What is the recombinant gene generated into?

A

into embryonic stem cells generated by transfecting DNA

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10
Q

What cells are injected into an embryo?

A

ES cells with proper gene structure

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11
Q

What are the steps to making the desired “knock out” mouse?

A
  1. Take stem cells out of mouse
  2. Add desired DNA to the cultured embryonic stem cells
  3. Select the cell the gets the proper homologous recombination (bc can possibly integrate desired change in wrong location) and expand
  4. Add selected cells to a blastocyst (early early embryo development stage) and implant the modified blastocysts back into the mouse, this mouse is called the “founder mouse”
  5. The founder mouse has the KO gene (one w/ proper recombo) in its germ cells.
  6. The founder mouse is then used to make mice heterozygous for desired genetic change
  7. The heterozygous generation is then bred until a homozygous mouse that displays the desired gene
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12
Q

What revelas locations of EVC gene?

A

expressionof the LacZ

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13
Q

What are 2 types of genetic engineering that are directly relevant to human medicine?

A
  • Genetically engineered cells, especially stem cells.
    Ex. Important in cancer research, etc.
  • Pharmaceuticals produced from genetically engineered bacteria.
    Ex. Insulin, etc.
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14
Q

What allows for site-specific DNA targeting?

A

Cas9 system

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15
Q

What is the Cas9 approach to genome editing?

A
  1. acts as endonuclease & cleaves DS DNA
  2. donor DNA added to match seqeunces
  3. homology directed repair occurs
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16
Q

What has a T-cell receptor that binds to a tumor antigen?

A

T-cell

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17
Q

How can the bacterial clone be amplified?

A

in a liquid culture

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18
Q

How can insulin be recovered?

A

from liquid media

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19
Q

What are hte 2 bacterial recombinant DNA treatments?

A

insulin & growth hormone

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20
Q

What is the process of humanizing antibodies?

A
  1. Take the Ag binding site from the rodent Ab
  2. Integrate the Ag binding site with the Ab sequence of humans
  3. Result is an Ab that is all human except for an Ag binding site from a rodent
21
Q

What are the recombinant Abs made by recombinant DNA cloning in autoimmune diseases?

A

TNF-alpha -> adalimumad (humira)

22
Q

What are the recombinant Abs made by recombinant DNA cloning in cancer?

A

VEGF -> bevacizumab (avastin)

23
Q

Where do we have stem cells?

A

in bone marrow

24
Q

How do stem cells go through self renewal?

A

When dividing one daughter cell will stay a stem cell, while the other differentiates

25
What are the best stem cells and why?
embryonic stem cells becuase they are potent
26
What are alloreactions?
bad reactions that occurs when the T-Cells of a person reacts to the HLA of another person
27
When does graft rejection happen?
if we do not have proper HLA matching or autologous cells.
28
What is cyclosporine?
an immunosuppressive medication given to prevent alloreaction & graft rejection
29
What are autologous cells?
are cells/desired structures obtained from using a patients own stem cells
30
What prevents immune reaction/rejection?
autologous cells
31
What is changing cell phenotype trandifferentiation?
causing expression of different genes to produce a different cell
32
What approaches strive for autologous tissue?
trans-differentiation, via transformation of cells with expression vectors for transcription factors
33
What undoes differentiation?
Vectors for Transcription Factors for dedifferentiation
34
What is the protein needed for dedifferentiation?
OCT 4 & SOX2
35
Where are hematopoietic stem cells from?
bone marrow
36
What do hematopoietic stem cells express?
CD34+ cell
37
What are the way of getting a stem cell?
* Using CD34+ blood cells (found in bone marrow) * Transdifferentiating w/ transcription factors
38
What are the 3 options of adding a new gene to a defective cell to try and save it?
* add a gene -> gene augmentation * silence a gene -> gene silencing * CRIPR/Cas 9 repair of mutant gene
39
What is needed for packaging the RNA into the caspid?
psi at the 5' end
40
What are the 2 viruses used as vectors?
* lentiviruses (HIV) * adeno-associated viruses (AAVs)
41
What is the gene defective in muscular dystrophy?
dystrophin
42
What are the 2 versions of muscular dystrophy?
* in frame deletion of multiple central exons -> mild -> Becker muscular dystrophy * out of frame deletion of central exon -> severe -> Duchenne muscular dystrophy
43
What is needed to blocking splicing of exon 51?
antisense oligonucleotide (AO)
44
From the reading, what holds potential for application as cellular therapy?
mesenchymal stromal cells
45
From the reading, what is immune rejection caused by?
human leukocyte antigen (HLA) mismatches
46
From the reading, what are the 2 methods used?
* knock out of the beta-2-microglobulin gene * fusion gene knocked in using the CRISPR/Cas9 system
47
From the reading, the B2M gene knock out protected cells from what but were made vulnerable to what?
protected cells from allogeneic T cell immune responses but were vulnerable to NK cells
48
From the reading, what protected cells from both T & NK cells?
B2M gene knock-out in combination with B2M-HLA-G knock-in

Genetics (11 decks)

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