ALAT Exam 1 Flashcards

Question

Adenoviral Infection

Answer 1

No integration, temporary expression, requires boost ## Footnote transient, in vacuole not nucleus

Answer 2

mouse and rat host

Answer 3

most mammals, including human hosts

Answer 4

The virus is cut into three parts, each having a LTR (long terminal repeat on each end); envelope, packaging, and transfer plasmid * Allows virus to enter cells in culture but make it extremely hard for recombination to occur and for the virus to become contagious * There is no replicating part so once the virus is in the cell it will not continue to replicate * The more elements that are on different plasmids the less risk to the population

Answer 5

Done through transfection Thaw competent cells and put on ice Aliquot cells and DNA into snap cap tubes, 1 tube/ plasmid Combine and heat shock - so DNA can enter the bacterial cells Plate bacteria onto LB agar + amp plates and allow to incubate until colonies form Place single colony into LB broth overnight Mini-Prep to collect the DNA and spec for amounts Combine DNA with serum free media Incubate cells with turbofect for 20 min Drop by drop add viral media to plate that was plated the night before with 4X106 cells Can analyze transfection rate using flow after 24-48 hours ## Footnote Do not want growth serum because you don’t want to kill cells or form toxic components

Answer 6

Internal Ribosomal Entry Sequence Allows for the creation of two separate proteins because the ribosomes can bind in the middle of the sequence, without these the two proteins would be fused together

Answer 7

Enable the expression of a specific gene or genes in a host organism, such as bacteria, yeast, or mammalian cells.

Answer 8

Creating an expression vector involves inserting a specific gene into a plasmid vector. This gene is then cloned into the vector using restriction enzymes and ligase. The newly formed plasmid is then introduced into a host organism (typically bacteria), for replication and protein expression Design (vector, PCR primers, etc.), PCR, Electrophoresis, Restriction Digest (specific enzymes to cut section you want to insert vector), gel purification, transformation, mini prep, sequencing

Answer 9

1. Self-sufficiency in growth signals 2. Insensitivity to antigrowth signals 3. Apoptosis Evasion 4. Limitless (immortal) replicative potential 5. Sustained angiogenesis 6. Tissue invasion and metastasis 7. Deregulating cellular energetics 8. Genome instability and mutation 9. Avoiding immune destruction 10. Tumor - promoting inflammation

Answer 10

Normal cells require growth cells to proliferate, but cancer cells escape this requirement Ligand independent growth Cycle faster → skip a lot of cycle checkpoints

Answer 11

Normal tissues are protected from overproliferation by a variety of inhibitory signals, but cancer cells are insensitive of these Ignores signals that inhibit cell growth

Answer 12

Apoptosis is used by normal cells to prevent damaged or defective cells from continuing to divide; apoptosis is inhibited or disrupted in cancer cells.

Answer 13

Normal cells have limited replicative potential due to telomere loss; cancer cells contain active telomerase (or other mechanisms) to maintain telomeres ## Footnote Some cell lines have this property to be able to continue a cell culture

Answer 14

(Solid) Tumor cells cannot grow beyond a few mm without a blood supply; cancer cells trigger angiogenesis by activating genes coding for angiogenesis stimulators and inhibiting genes coding for angiogenesis inhibitors ## Footnote In large tumors, the middle is usually necrotic due to lack of blood supply

Answer 15

Cancer cells lose adhesiveness with neighbors, invade nearby tissues, and eventually metastasize around the body via the circulatory system Tissue invasion: invades local tissue but hasn't moved out Metastasis: cancer has moved out of tissue of origin

Answer 16

Tumor cells are shown to reprogram cellular metabolism in order to support neoplastic proliferation More metabolically active PET Scan detects unexpected loss of weight

Answer 17

Increased mutability endows cancer cells with alterations that drive tumor progression The more the tumor divides, the more mutations occur Check points are bypassed Knowing which oncogenes the tumor needs, which have no effect, which harm it, etc. help determine best course of treatment

Answer 18

Evasion of the immune system and destruction of cancer cells Immunotherapy

Answer 19

It has been shown that inflammatory responses can be tumor-promoting environments Regulatory T cells down regulate T cells and can inhibit immunotherapy Obese → ↑ inflammation → ↑ risk of cancer

Answer 20

cancer cells lose contact inhibition and will grow on top of one another Normal cells grow in a monolayer Often due to the down regulation of integrins so they don’t have to fully flatten and attach before division (cells will show as clumps in tissue culture) Don't attach as well to flask/plate

Answer 21

Bottom layer is harder agar which prevents the cells from attaching to the plate, tumor forms in soft layer, non cancerous cells are suspended and die Prevents normal cells from growing by inhibiting contact Feeder layer above cell-containing soft layer

Answer 22

Human cells are put into immunocompromised mice Hairless mice make it easier for researcher can see the growth of the tumor Generally injected into the back flank so the mouse can’t chew on the area (if something happens where the tumor breaks through it must be euthanized)

Answer 23

Splenomegaly, enlarged thyroid, lymph nodes, and liver, due to the undifferentiated T cells being stuck in the organs in the periphery CD4 and CD8 double positive cells will be out in the periphery

Answer 24

NotcIC induces T-ALL in mice when expressed in pre-T cells or bone marrow Mutations found in HD region (embeds notch in membrane) an PEST domain (involved in stability of proteins) When not working properly the increased expression of MYC will not induce p53, however the main mutation is with notch because in MYC driven tumors there is still 20% relapse

Answer 25

an increased expression will block p53 expression, but when there is an increase in notch, MYC will increase in expression and be able to induce p53

Answer 26

the increased expression of MYC will not induce p53, however the main mutation is with notch because in MYC driven tumors there is still 20% relapse

Answer 27

Day before plate 6 wells with 5X105 cells per plate, allow to incubate overnight Remove media from plate and perform the following serial dilution, these numbers can be changed but need a 1:10 dilution, when aliquoted 1.2 ml is frozen down * Well 1: 1.2 ml viral media * Well 2: 0.2 ml viral media from above and 1.8 ml normal media * Well 3: 0.2 ml viral media from above and 1.8 ml normal media * Well 4: 0.2 ml viral media from above and 1.8 ml normal media * Well 5: 0.2 ml viral media from above and 1.8 ml normal media * Well 6: 1 ml normal media Only need to titer one per viral prep because they should all be the same After 4 hrs add 2 ml of normal media and collect cells after 48 hours to evaluate

Answer 28

when looking at infection curve want to choose a value in the middle, ensures that the number are starting to plateau, after you get numbers do the following calculations (ml/%) x [X(ml solving for)/% you want to infect] → solve for X to know how much virus to use Also do not want 100% efficiency because that would not even be possible for bone marrow stem cells, can change based on the cells that you will be infecting

ALAT Exam 1 Flashcards

(52 cards)