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Reverse Genetics:
interference RNA
Mitchell Bush
Mimosa Chau
Tayler Fluharty
Summary
• Use RNAi to circumvent gene replacement
difficulties in Cryptococcus neoformans.
• Focus on two genes of known phenotype:
CAP59
Codes for product required for
polysaccharide capsule.
Polysaccharide capsule required
for virulence.
ADE2
Codes for
phosphoribosylaminoimidazole
carboxylase.
Loss of AIR carboxylase results in
adenine intermediates.
General Mechanism of RNAi
Double stranded RNA is cleaved
into smaller fragments of about
22 bp by Dicer where
degradation of corresponding
mRNA is degraded with
assistance of RISC protein
complex.
Interference can persist for many
round of cell division and can be
passed on to next generation.
http://nobelprize.org/nobel_prizes/medicine/laureates/20
06/adv.html
Cryptococcus neoformans
• Encapsulated fungal pathogen.
• Haploid yeast, can undergo sexual cycle
• Causes infections in immunodeficient
individuals.
• 24 Mb genome.
• High frequency of nonhomologous
recombination.
• Virulence factor = polysaccharides
capsule.
Cell strains
• Wild-type serotype D strain B4500
(transformed with control plasmid lacking RNAi construct)
• cap59 strain TYCC33
(CAP59 knocked-out/positive control)
• ura5 strain JEC43
(used for two type of selection during experiment)
Hairpin Construct
• 500 base Inverted Repeat(s)
• 250 base spacer of GFP
CAP59 encodes product required for
synthesis of polysaccharide capsule
which gives cells a “shiny” appearance
CAP59i cells lack polysaccharide capsule
(note exception)
ADE2 encodes for
phosphoribosylaminoimidazole
carboxylase
ADE2i cells accumulate adenine
biosynthetic intermediates,
resulting in pink colonies.
DIC
Fluorescence
DIC
Fluorescence
5-FOA is converted to toxic product in presence of RNAi
construct (pCAP59i/ pADE2i)
Therefore 5-FOA selects against organisms containing the RNAi
construct.
Mutant phenotype caused by exogenous DNA
The ADE2 gene in the genome was not disrupted.
RNAi is dependent on
Transcription
Made plasmid that lacked promoter for
CAP59i.
No transcription=No altered phenotype
Summary of Results
• The effect of RNAi is variable.
• Different genes have different threshold for
phenotypic expression.
• CAP59 has a lower threshold than ADE2
Range of interference
Pro
 study of essential
genes-if complete
inhibition were lethal
Con
 Investigation of novel
genes requires 2phases of study: level
of mRNA and analysis
of transformants
exhibiting interference.
In the Works
Fight a virus with RNAi:
Foot and mouth disease virus (FMDV)
• Data base search for highly conserved regions of
FMDVThree 21 base sequences found.
• Silence out one at a time, then all three
Viral inhibition (%)
Cons 7 80
Cons 8 92
Cons 9 87
mix
>98
Three potential
mechanisms to fight
HIV:
1) CD4 silencing resulted in
reduced HIV-1 entry, but
use may be limited due to
normal function of CD4
2) Blocking HIV genomic
RNA with p24 siRNA
resulted in reduced viral
mRNA (CCR5 is potential
future target lack of this
receptor does not disrupt
regular cell function)
3) Blocking HIV transcript
with p24 siRNA inhibits HIV1 replication
RNAi and cancer
• Metastatic melanoma – Genasense
• Antisense oligonucleotide target antiapoptotic gene BCL2.
• Bcr-Abl oncoprotein (chronic myelogenous
leukaemia ) p210 targeted with RNAi.
• Delivery proves to be a challenge.
• Delivery agents such as cationic lipids or
polymers. Problem = toxic.
Delivery methods
• Oligonucleotides/siRNA delivered via
endocytosis.
• CTLs, cell targeting ligands – increases target
cell interactions.
• CPP primarily cell penetrating peptides enhance transmembrane permeation –
associate with oligonucletide, binds to cell
surface glycosaminoglycans.
“Significant strides have been made, but the
issue has not been fully resolved.”
-Juliano, Et. al
References
Daneholt, B. (2006). The Nobel Prize in Physiology or Medicine 2006. Retrieved March 8, 2009,
from Nobelprize.org: http://nobelprize.org/nobel_prizes/medicine/laureates/2006/adv.html
Hannon, G. J., & Rossi, J. J. (2004). Unlocking the potential of the human genome with RNA
interference. Nature , 371-378.
Juliano, R., Alam, R., Dixit, V., & Kang, H. (2008). Mechanisms and strategies for effective
delivery of antisense and siRNA oligonucleotides. Nucleic Acids Research , 1-14.
Kahana, R., Kuznetzova, L., Rogel, A., Shemes, M., Hai, D., Yadin, H., et al. (2004). Inhibition of
foot-and-mouth disease virus replication by smal interfering RNA. Journal of General Virology ,
3212-3217.
Liu, H., Cottrell, T. R., Pierini, L. M., Goldman, W. E., & Doering, T. L. (2001). RNA Interference in
the Pathogenic Fungus Cryptococcus neoformans. Genetics Society of America , 463-470.
Novina, C. D., Murray, M. F., Dykxhoorn, D. M., Beresford, P. J., Riess, J., Lee, S.-K., et al. (2002).
siRNA-directed inhibition of HIV-1 infection. Nature Medicine , 681-686.
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