Plasmodium falciparum Parasites Oct/28/08 Feng, Chi-wei Supervised - - PowerPoint PPT Presentation

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Plasmodium falciparum Parasites Oct/28/08 Feng, Chi-wei Supervised - - PowerPoint PPT Presentation

Dec 11, 2023 116 likes •411 views

1 A Regulatable Transgene Expression System for Cultured Plasmodium falciparum Parasites Oct/28/08 Feng, Chi-wei Supervised by Prof. Ching-Tsan Huang, PhD 2 Plasmodium falciparum 8~24 merozoits rupture By the courtesy of Bannister, L. H.

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A Regulatable Transgene Expression System for Cultured Plasmodium falciparum Parasites

Oct/28/08

Feng, Chi-wei

Supervised by Prof. Ching-Tsan Huang, PhD

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Plasmodium falciparum

By the courtesy of Bannister, L. H. et al. and Stockbower, R.

2 8~24 merozoits rupture

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The Life Cycle of Human Malaria Parasite, Plasmodium falciparum

By Courtesy of Brian M. Greenwood et al., “Malaria: progress, perils, and prospects for eradication” , The Journal of Clinical Investigation, Vol.112, Number 4, April2008

Erythrocytic cycle

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Blood Stages of P. falciparum

  • 1. Ring
  • 2. Trophozoite
  • 3. Schizont

Multiple invasion

By Courtesy

  • f Dr. Chairat

Uthaipibull

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Current developments

By courtesy of Y Wu. et al

Annotated genome data base Gene knockout technique by Homologous Recombination

By courtesy of Daily JP. et al

Protein overexpression 5

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The 2 shortcomings

  • The oversized episome with AT-rich

sequence is not favorable by E.coli in multiplication.

  • Limited copy number of episome and low

efficiency of promoter.

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Previous efforts

  • P.falciparum gene expression regulation by:

the Atc-inducible system, FKBP12 system, use of ribozyme actions

  • Complexity
  • f system in manipulation
  • Narrow utility that’s hardly widespread

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Strategy

Bidirectional Promoter Selectable marker Drives both selectable marker and Transgene Regulation of expression by concentration

  • f drug

A regulatable expression system by a mini-sized plasmid

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General plasmid with promoter

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(1 Gene+1 Promoter)x N= Plasmid in large size

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Bidirectional Promoters

  • A series of gene promoting and silencing

experiments in studies in genes of interested in Plasmodium species

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  • PFC0005w
  • PFB1055c
  • PFD0020c

Introns with promoter activity in Bidirectional manner

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  • 1. Stable transgenic construct

in minimized size

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Trangenic Construct- pHLIDH

The VLH region was replaced by HL

pHLIDH

PstI KpnI 12

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Trangenic Construct- pHLIDH

(H: hrp2 3’UTR, L: firefly luciferase )

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Promoter activities of the Introns

The intron PFC0005w shows the highest promotion activity according to luciferase activity among the three. Luminescence was expressed per 1% parasitaemia 14

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Coordination in promoting actitivity

RT-PCR pHLIDH with PFC0005w is suitable for late gene expression : luciferase : dfhr : spb1 : msp1 15

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  • 2. Regulation in transgenic

expression level

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Trangenic Construct- pHBIRH

(H: hrp2 3’UTR, B: blasticidin-s-deaminase; bsd, R: Renilla luciferase)

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Blasticidin-s

Formula: C17 H26 N8 O5 , Aminohydrolase substrate Isolated from Streptomyces griseochromogenes

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Promoter activities of the Introns

The promoter PFC0005w shows the highest activity according to luciferase activity among the two. 19

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Luciferase activity vs. Drug conc.

Luciferase expression is stage-specific, and yet coordinate to drug concentration. Luminescence was expressed per 1% parasitaemia 20 : 5 μg/ml : 2 μg/ml : 10 μg/ml : 20 μg/ml Blasticidin-s conc. Increase in drug concentration

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Increase in Copy number

By DNA extraction, Increase in bsd and Renilla luciferase expression is due to change in copy number of construct. 21 : 5 μg/ml : 2 μg/ml : 10 μg/ml : 20 μg/ml Blasticidin-s conc.

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Increase in gene expression

The relative amount of bsd and Renilla luciferase mRNA directly correlated with blasticidin concentration in the late stage. 22 : 5 μg/ml : 2 μg/ml : 10 μg/ml : 20 μg/ml Blasticidin-s conc.

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Conclusion- Pros and Cons

  • Transgene expression will be brought about due

to bsd expression, which give rise to ability of regulation.

  • Simplicity and stability as a bidirectional

construct in smaller size.

  • Applicability in Protein Localization studies
  • Adjustment of the level of gene expression only

by copy number. Cannot be switched off completely.

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24 Bacterial Plasmid P.falciparum system: Oversize and Complexity Bidirectional Activity Regulation in gene expression level

Protein over-expression, trafficking in the parasite, etc

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Thank you for your Attention! Special Gratitude to

  • Prof. HUANG

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Definition

  • Parasitaemia: the ratio of a group of RBC being

invaded be parasite(s). Regular ratio~1%

  • eg. A group of 1000 rbc within which 20 are

invaded yields parasitaemia of 2%.

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Problems in previous efforts

  • ATc-inducible system: tetracycline analogue called
  • anhydrotetracycline. This system’s regulatable in blood

stages.

✖Tedious in 2 different selection cycles ✖Very large in Construct size

  • FKBP12 system: Engineered destabilizing domain of

human FK506 binding protein (ddFKBP), promoting degradation of fused protein. But in the presence of Shld1 , degradation is reduced.

✖Efficacy is limited to the protein investigated

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Blasticidin-s deaminase

  • Blasticidin-S aminohydrolase
  • EC 3.5.4.23 PDB access: 1WN5/1WN6
  • Blasticidin-S + H2

O = deaminohydroxyblasticidin-S + NH3

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