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Biol 432 > Midterm 1 > Flashcards

Midterm 1 Flashcards

lectures 1-7

1
Q

gibson assembly of fragments

A
  • take 2 blunt ends –> PCR amplify using primers to create overlapping regions
  • amplicons have overlap –> use matermix enzymes to trim down overlaps –> overhangs
  • annealing of overhangs
  • DNA pol + ligase to fill gaps + nicks
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2
Q

CRISPR acronym

A

clustered regularly interspaced short palindromic repeats

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3
Q

crispr gene editting requirements

A

target sequence
chimeric gRNA (simplification fulfilling crRNA and trRNA fxns)
CAS9 endonuclease (induce DSB)
ssDNA (specific desired mutation flanked by homologous regions –> allow HDR)
PAM site (distinguishes target seq vs bacterial crispr array)

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4
Q

HDR vs NHEJ

A

NHEJ = randomized indels

HDR = controlled, specified, indels

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5
Q

talens/Zn fingers

A

peptides that bind to target
DNA binding domains flank site of mutagenesis
linked to FOK1 nucleases –> cover site of mutagenesis
rec domains MUST perfectly overlap –> +/- 1 nt =/= DSB

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6
Q

CRISPRi - gene silencing

A

inactivated CAS9 –> just sits on the GOI –> blocks RNA Pol –> gene is silenced

remove Cas9 –> gene is reactivated

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7
Q

CRISPR CF1 modification

A
  • use 2 gRNAs –> induce ssDNA nicks in CLOSE proxy –> allows HDR indels
  • nicks far apart –> no HDR indel –> independent nicks easily repaired w/o issues –> decreases crispr off-targeting errors
  • chances of nicks in close proxy = low –> increased crispr specificity
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8
Q

Interferon for Protein Therapeutics

A
  • Interferons (IFN gene) –> expressed cytokines to alert immune response –> increased efficiency of treatments
  • Effectiveness can be further improved using gene shuffled IFN variants –> maximize interferon effectiveness –> combine IFN alpha 1 and alpha 2
  • issue with interferons: hard to purify
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9
Q

half life of protein therapeutics

A
  • time for [proteins] to halve
  • can be improved using polyethylene glycol (PEG) –> pegylation
  • PEG improves protein stability + decreases excretion from body (persists in body longer)
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10
Q

examples of extending half life of protein therapies (alternatives to PEG)

A

HGH, XTEN protein tag

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11
Q

Extending half life - HGH

A

modify HGH w/receptor peptide –> causes HGH dimerization
dimerized –> harder to degrade –> increased half life

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12
Q

Extending half life - XTEN tag

A

Alternatives to Pegylation
protein tag increases stability –> higher half life
allows purification of product using CBD
tag is cleaved off of product to obtain fxnal form

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13
Q

Combating cystic fibrosis

A
  • caused by P. aeruginosa –> forms alginate biofilm –> difficult to deliver drugs
  • Soln 1: introduce alginate lyase produced/harvested from Ecoli to degrade biofilm. Bacterially derived enzyme –> immunogenic. Mitigate by mutating epitopes
  • Soln 2: use non lytic phage M13 carrying LexA gene. non lytic –> minimal pressure against the phage. LexA interrupts SOS DNA repair –> Become susceptible to quinalone treatments
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14
Q

Crohn’s/Ulcerative colitis treatment

A
  • Delivery of drugs via LABs native to gut microbiome
  • integrate interleukin 10 (IL-10) to become thymidylate synthase (-) –> can’t synthesize thymidine –> reliant on patient to scavenge
  • integration of IL-10 ensures modified bacteria cannot persist in nature
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15
Q

AIDs treatment

A
  • Cyanovirin N expression using L. bacillis (native to vagina)
  • constitutive expression of Cyanovirin N –> binds AIDs gp120 and gp41 receptors –> blocks infection
  • improve cyanovirin N protein stability by replacing Gly w/ Pro
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16
Q

Antibody stuff definitions

A

antigen (Ag) = foreign object
antibody (Ab) = proteins from plasma cells to facilitate epitome-macrophage interactions
epitope = specific structure on the Ag recognized by Ab

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17
Q

antibody structures

A

heavy chain + light chain
FC (constant, crystalized) region = leukocyte intrx domain
Fab = epitope recognition region
FV (variable) = specific portion of Fab for epitope recognition

18
Q

harvesting mAb

A
  • inject Ag to mouse –> pAb excreted
  • 2 injections to ensure high Ab specificity
  • harvest plasma cells from spleen –> fusion by PEG w/HGPRT(-) myeloma
  • selection on HAT media for immortalized B cell hybridoma
  • dilute to isolate 1 cell/drop –> selection for mAb to target epitope using ELISA
  • culture and express mAb of interest
19
Q

Ab humanization

A

purpose of humanization = decrease risk of immunogenic response from Ab treatments
- use Fab with human Fab constant region + mouse FC –> partial humanization
- engineer human Ab with FC containing mouse CDR regions –> 95% humanized

OR make humanized mouse
- lineage 1 = delete both IgH and IgK heavy/light chain genes
- lineag2 2 = introduce human IgH and Igk
- crossbreed –> select offspring mice carrying only humanized IgH and Igk –> xenomouse –> produces only human antibodies

20
Q

optimizing Ab

A

creation of partial Ab’s
- FC = actual epitope recognition –> dont need the Fab constant domain –> isolate FC
- link 2 using linkage peptide FC –> diabody
- 2 of same FC –> bivalent diabody
- 2 of different FC –> bispecific diabody

21
Q

example usage of diabodies

A
  • use Irinonectan on cancer cells –> induce cancer-specific cell receptors
  • use bivalent diabody. FC1 binds receptor, FC2 binds/carries a toxin –> targetted delivery of toxin to cancer cells
22
Q

Abzymes

A

Ab with catalytic FC –> abzyme

23
Q

example abzyme usage

A

more ghrelin protein = more hunger
- inject transition state analogue to ghrelin –> mouse excretes enzymatic Ab –> able to cleave ghrelin precursor
- therefore prevent hunger accumulation

24
Q

antisense RNA

A
  • antisense cDNA –> antisense RNA –> bind transcript –> block expression
  • RNA oligonucleotide –> bind transcript –> block expression
25
Q

improving antisense oligonucleotides

A
  • Replace P with S in phosphatediester bonds –> resistant to nuclease degradation
  • capping with methoxyethyl –> resist nucleases + higher target affinity
  • introduce deoxynucleotides to form RNA-DNA duplex –> activates RNAseH –> Cleave target transcript
26
Q

Retroviral life cycle

A
  • retroviral RNA –> reverse transcriptase to form RNA-DNA duplex
  • RNA strand degraded –> cDNA used to form retroviral dsDNA
  • retroviral DNA integrated into bacterial genome for replication
27
Q

Interrupting retroviral life cycle using DNA therapeutics

A

Introduce oligonucleotide to occupy retroviral tRNA site –> block synthesis of cDNA. Therefore no retroviral DNA is synthesized

28
Q

RNA Aptamers

A
  • Similar to Ab’s –> high affinity for target molecule (protein or other NAs)
  • unlike AB’s –> NOT immunogenic (body doesnt react to NAs, only proteins)
29
Q

creation of aptamers

A

SELEX selection
- random nucleotide sequences –> express random RNA aptamers
- test on epitope of interest –> find aptamer that successfully binds
- dissociate bound aptamer –> cDNA –> find sequence of aptamer
- PCR the apamter cDNA –> rescreen on epitope –> repeat selection to increase affinity

30
Q

Ribozymes

A

Recall: RNA can be enzymatic
- recognition domain to recognize with substrate
- enzymatic domain

31
Q

RNAi - RISC complex

A
  • introduce dsRNA (or vector expresses hairpin RNA shRNA)
  • DICER enzyme cleaves dsRNA –> siRNA
  • Argonaute enzyme binds siRNA –> remove passenger RNA –> ssRNA
  • Argonaute + recognition ssRNA = RISC complex
  • RISC binds target transcript using ssRNA hybridization –> cleave transcript

results in ~90% reduction to expression

32
Q

Viral methods of NA delivery

A

RHAL
- retrovirus - encode GOI in retroviral genome –> transform –> GOI as cDNA –> integrate
- lentivirus - removal of nonpathogenic genes –> use to infect non-dividing cells
- adeno-associated virus - TINY virus, relies on helper adeno virus to replicate. Deliver alongside adeno –> replication. No adeno –> integration of GOI
- HSV1 - can carry up to 150 kb of nt –> MASSIVE gene delivery

33
Q

Viral delivery of NAs - SCIDS

A
  • rare disease - T cell deficiency
  • originally treat by using pegylated adeno deaminase (ADA) - ADA stimulates T cells
  • PEG causes immunogenic response –> ADA treatment only works short term
  • deliver wt IGL2 gene (to repalce mutated ILG2) using retrovirus –> hematopoetic stem cells –> transplant
  • retrovirus modified to prevent leukemia side effect
34
Q

NON viral methods of GOI delivery - non carrier molecules

A

LMB-Z
- modified zygote carrying GOI –> transplant to surrogate
- liposome nanoparticle - modified with lipoproteins to carry NAs inside
- Minicells - transform with GOI into mutated bacteria–> replicates as minicells –> act as naturally derived liposomes (non immunogenic)
- non pathogenic bacteria - GOI in plasmid + invasin –> invasin gene allows interaction with mammalian B1-integrin receptors

35
Q

carrier molecules for delivery of GOI

A

DAAC
- Aptamer
- single chain antibody - FV to recognize target host receptor. FC linked with (+) protoamine (attracts GOI)
- Dendrimer - large non immunogenic molecule (+) charged (carries the GOI) –> endocytosis
- siRNA (for RISC) linked to cholesterol

36
Q

siRNA - cholesterol delivery - ApoB gene

A

targetted delivery of siRNA using cholesterol carrier to target ApoB gene –> 50-70% reduction of serum cholesterol

37
Q

traditional vaccines

A

heat killed/inactivated virus –> decrease virulence but maintain immunogenicity
attenuated virus –> pass virus through animal –> mutation reduces virulence to humans

limitations: expensive + low product yields, hazardous to culture, not all virusses can be easily cultured, attenuated virus can revert back to regain human virulence

38
Q

subunit vaccine

A

overexpression of a speciifc peptide (subunit) epitope to illicit immunigenic response

HIGHLY SPECIFIC to that particular epitope

39
Q

peptide vaccine

A

several subunits/fragments linked to a carrier protein (BSA or KLH) –> subunits alone = too small to be recognized by immune system

can use multiple diff subunits to create vaccine for multiple antigenic sites

40
Q

DNA vector vaccine

A

encode viral DNA or cDNA in plasmid + CpG adjuvant gene (adj stimulates immune response) + polyG tail to increase uptake of vaccine

plasmid delivered to host –> expression of epitopes/antigenic fragments –> present on MHCI –> T cells respond –> formation of memory cells + antibodies

41
Q

MIDGE vector vaccine

A

DNA vector vaccine is cloned using antibiotic selectable marker –> not ideal for human usage –> needs to be removed

excise GOI and cap ends to prevent degradation –> MIDGE vector

Biol 432 (3 decks)

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