Genetic Engineering

Question 1

What are the 6 types of gene therapies?

Answer 1

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

Question 2

What are 2 ways to add exo-siRNA?

Answer 2

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

Question 3

What is transfection?

Answer 3

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

Question 4

What is transformation?

Answer 4

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

Question 5

What does shRNAi lead to?

Answer 5

suppression of transcription

Question 6

What is associated with dysplasia?

Answer 6

knock out mouse lacking EVC cells

Question 7

What do gene targeting vectors replace?

Answer 7

exons

Question 8

What is meant by the term “knock in”?

Answer 8

Put something in place of the endogenous gene

Question 9

What is the recombinant gene generated into?

Answer 9

into embryonic stem cells generated by transfecting DNA

Question 10

What cells are injected into an embryo?

Answer 10

ES cells with proper gene structure

Question 11

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

Answer 11

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

Question 12

What revelas locations of EVC gene?

Answer 12

expressionof the LacZ

Question 13

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

Answer 13

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

Question 14

What allows for site-specific DNA targeting?

Answer 14

Cas9 system

Question 15

What is the Cas9 approach to genome editing?

Answer 15

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

Question 16

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

Answer 16

T-cell

Question 17

How can the bacterial clone be amplified?

Answer 17

in a liquid culture

Question 18

How can insulin be recovered?

Answer 18

from liquid media

Question 19

What are hte 2 bacterial recombinant DNA treatments?

Answer 19

insulin & growth hormone

Question 20

What is the process of humanizing antibodies?

Answer 20

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

Question 21

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

Answer 21

TNF-alpha -> adalimumad (humira)

Question 22

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

Answer 22

VEGF -> bevacizumab (avastin)

Question 23

Where do we have stem cells?

Answer 23

in bone marrow

Question 24

How do stem cells go through self renewal?

Answer 24

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

Question 25

What are the best stem cells and why?

Answer 25

embryonic stem cells becuase they are potent

Question 26

What are alloreactions?

Answer 26

bad reactions that occurs when the T-Cells of a person reacts to the HLA of another person

Question 27

When does graft rejection happen?

Answer 27

if we do not have proper HLA matching or autologous cells.

Question 28

What is cyclosporine?

Answer 28

an immunosuppressive medication given to prevent alloreaction & graft rejection

Question 29

What are autologous cells?

Answer 29

are cells/desired structures obtained from using a patients own stem cells

Question 30

What prevents immune reaction/rejection?

Answer 30

autologous cells

Question 31

What is changing cell phenotype trandifferentiation?

Answer 31

causing expression of different genes to produce a different cell

Question 32

What approaches strive for autologous tissue?

Answer 32

trans-differentiation, via transformation of cells with expression vectors for transcription factors

Question 33

What undoes differentiation?

Answer 33

Vectors for Transcription Factors for dedifferentiation

Question 34

What is the protein needed for dedifferentiation?

Answer 34

OCT 4 & SOX2

Question 35

Where are hematopoietic stem cells from?

Answer 35

bone marrow

Question 36

What do hematopoietic stem cells express?

Answer 36

CD34+ cell

Question 37

What are the way of getting a stem cell?

Answer 37

* Using CD34+ blood cells (found in bone marrow) * Transdifferentiating w/ transcription factors

Question 38

What are the 3 options of adding a new gene to a defective cell to try and save it?

Answer 38

* add a gene -> gene augmentation * silence a gene -> gene silencing * CRIPR/Cas 9 repair of mutant gene

Question 39

What is needed for packaging the RNA into the caspid?

Answer 39

psi at the 5' end

Question 40

What are the 2 viruses used as vectors?

Answer 40

* lentiviruses (HIV) * adeno-associated viruses (AAVs)

Question 41

What is the gene defective in muscular dystrophy?

Answer 41

dystrophin

Question 42

What are the 2 versions of muscular dystrophy?

Answer 42

* in frame deletion of multiple central exons -> mild -> Becker muscular dystrophy * out of frame deletion of central exon -> severe -> Duchenne muscular dystrophy

Question 43

What is needed to blocking splicing of exon 51?

Answer 43

antisense oligonucleotide (AO)

Question 44

From the reading, what holds potential for application as cellular therapy?

Answer 44

mesenchymal stromal cells

Question 45

From the reading, what is immune rejection caused by?

Answer 45

human leukocyte antigen (HLA) mismatches

Question 46

From the reading, what are the 2 methods used?

Answer 46

* knock out of the beta-2-microglobulin gene * fusion gene knocked in using the CRISPR/Cas9 system

Question 47

From the reading, the B2M gene knock out protected cells from what but were made vulnerable to what?

Answer 47

protected cells from allogeneic T cell immune responses but were vulnerable to NK cells

Question 48

From the reading, what protected cells from both T & NK cells?

Answer 48

B2M gene knock-out in combination with B2M-HLA-G knock-in