danijela research update
DESCRIPTION
Danijela Lab Meeting Presentation PDFTRANSCRIPT
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Research Update: Iden0fying and Characterizing First Infected Cells in the Rectum
Danijela Marić March 11, 2015
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Schema0c Representa0on of Rectal Tissue
Nature reviews
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Research Goals
• To detect transduced cell foci in the rectal 0ssue using In Vivo Imaging System (IVIS)
• To iden0fy transduced cells in 0ssue sec0ons and validate them using an0body staining and Spectral Imaging
• To understand how viral envelopes determine target cell tropism in the rectal 0ssue
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Research Design • Lich Genera0on 2 reporter virus containing Luciferase and iRFP670
reporters was used • Several biopsies in the rectal compartment were introduced at
random • Female Rhesus Macaques were rectally challenged with Lich virus
containing either JR-‐FL or JR-‐CSF envelope • Forty-‐eight hours post challenge animals were sacrificed, recta
were removed and shipped to NU on ice overnight • Tissue was imaged using IVIS before and aWer addi0on of luciferin
to establish the level of background luminescence in the 0ssue • Areas with persistent luciferase signal post luciferin were cut out
and preserved at -‐80 degrees C in OCT for subsequent sec0oning, staining and imaging
• The infected cells are validated by an0body staining, spectral imaging and nested PCR and will be subsequently phenotyped
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Infec0on of TZM-‐bl Cells with pLi670 VSVg Virus
iRF670 bleed through in mCherry is minimal
iRFP670
iRFP702
iRFP720
iRFP670 iRFP702Mock iRFP720
Relative brightness of iRFPs detected in Cy5 channel, Jake.
10x
40x
Ex/Em
Spectral Profile of pLi670 Transduced Cell
Wes Grimm
LTR CMV iRFP670 WPRE LTR IRES Luciferase
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Research Goals
• To detect transduced cell foci in the rectal 0ssue using In Vivo Imaging System (IVIS)
• To iden0fy transduced cells in 0ssue sec0ons and validate them using an0body staining and Spectral Imaging
• To understand how viral envelopes determine target cell tropism in the rectal 0ssue
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Tissue Analysis Using IVIS RM HP63 (JR-‐FL)
PBS HP63_ 2 HP63_ 1
HP63_ 5 HP63_ 4 HP63_ 3
HP63_ 6
HP63_ Biopsy
Luciferin
HP63_ 5C HP63_ 5D HP63_ 5F
HP63_ 5E HP63_ 5A HP63_ 5B
HP63_ 6A
HP63_ 6B
HP63_ 6C
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Tissue Analysis Using IVIS RM GG70 (JR-‐FL)
GG70_ 6C GG70_ 6D GG70_ 6A
GG70_ 6B GG70_ 5 reimaged
PBS
GG70_ 1 GG70_ 2
GG70_ 3 GG70_ 6 GG70_ 5
GG70_ Biopsy
GG70_ 4
Luciferin
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Tissue Analysis Using IVIS RM HP63 (JR-‐CSF)
PBS FN94_4 FN94_1
FN94_2 FN94_6 FN94_ 3
FN94_5
FN94_ Biopsy
Luciferin
FN94_ 2A FN94_2B FN94_2E
FN94_2F FN94_2D FN94_2C
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Research Goals
• To detect transduced cell foci in the rectal 0ssue using In Vivo Imaging System (IVIS)
• To iden0fy transduced cells in 0ssue sec0ons and validate them using an0body staining and Spectral Imaging
• To understand how viral envelopes determine target cell tropism in the rectal 0ssue
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RM HP6 Piece 6A
RFP 670 (Direct Fluorescence) DAPI
The infected cell is in epithelia: Flower like ring structures of the rectal 0ssue are pointed by yellow arrows
IVIS: HP63_ 6A
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RM HP6 Piece 6A: Volume View Movie
RFP 670 (Direct Fluorescence) DAPI
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RM HP6 Piece 6A: Cell 1
RFP 670 (Direct Fluorescence) DAPI
Luciferase:FITC DAPI
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iRFP 670 Spectral Profile: Expected Maximum Emission: 670nm Observed Maximum Emission: 669nm
FITC Spectral Profile: Expected Maximum Emission: 519nm Observed Maximum Emission: 519nm
RM HP6 Piece 6A: Spectral Profile
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RM HP6 Piece 6A: More Examples of Infected Cells
RFP 670 (Direct Fluorescence) DAPI
RFP 670 (Direct Fluorescence) DAPI
Luciferase:FITC DAPI
Luciferase:FITC DAPI
RFP 670 (Direct Fluorescence) Luciferase:FITC DAPI
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GG70 Rectal Biopsy: Cell 1
iRFP670 (direct fluorescence) DAPI
iRFP670 (direct fluorescence) Luciferase:TRITC DAPI
Luciferase:TRITC DAPI
IVIS: GG70_ Biopsy
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GG70 Rectal Biopsy: Spectral Profile
iRFP 670 Spectral Profile: Expected Maximum Emission: 670nm Observed Maximum Emission: 668nm
FITC Spectral Profile: Expected Maximum Emission: 519nm Observed Maximum Emission: 520nm
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Research Goals
• To detect transduced cell foci in the rectal 0ssue using In Vivo Imaging System (IVIS)
• To iden0fy transduced cells in 0ssue sec0ons and validate them using an0body staining and Spectral Imaging
• To understand how viral envelopes determine target cell tropism in the rectal 0ssue
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Cell Phenotyping • Phenotyping transduced cells in the 0ssue by staining for various
cell surface markers (CD3, CD4, CD68, Th17,etc) • Op0mized 0ssue diges0on protocol involving collagenase
treatment and mechanical diges0on • Successfully isolated cells from rectal 0ssue (1x1 cm piece of 0ssue
yields ~20 million of cells, up to half were CD4+ T cells) • Op0mized freezing and storing protocol for the primary cells (over
90% cell viability was achieved post freezing and storing at -‐80 degrees C, 3 months later)
• Infec0on of rectal cells with mCherry Lich virus resulted in ~1% cells expressing mCherry protein
• Isolate RNA from the cells and perform RNA sequencing to establish the nature of the infected cells (in future)
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Building the Beier Reporter Constructs..
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Genera0on of Lich Gen3 HSA
• Use HSA tag to sort for the transduced cells isolated from the 0ssue
• Direct mCherry to the membrane so that localiza0on of Luciferase and mCherry would be dis0nct
LTR CMV mCherry WPRE LTR HSA paGFP Luciferase IRES
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293T Cells Transfected with GenX DNA Western Blot Analysis
An0-‐HSA WB
25 kDa
50 kDa
75 kDa
37 kDa
HSA:mCherry:GFP (70kDa)
Expected Sizes: HSA: 15 kDa mCherry: 27 kDa paGFP: 28 kDa
HSA/mCherry Luciferase
DAPI
293T Cells Transduced with GenX VSVg Virus Immunofluorescence Analysis
Expected Localiza0on: HSA and mCherry: Membrane Luciferase: Cytosolic
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Genera0on of Lich GenX
LTR CMV mCherry WPRE LTR HSA Luciferase
• Use HSA tag to sort for the transduced cells isolated from the 0ssue
• Ensure that fluorescent protein and Luciferase expression go hand in hand
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293T Cells Transfected with GenX DNA Western Blot Analysis
An0-‐mCherry An0-‐HSA An0-‐Luciferase
25 kDa
37 kDa
50 kDa
75 kDa
25 kDa
37 kDa
50 kDa
75 kDa
25 kDa
37 kDa
50 kDa
75 kDa 100 kDa
Expected Sizes: HSA: 15 kDa mCherry: 27 kDa Luciferase: 50 kDa
mCherry
mCherry/HSA Lucifearase/HSA
Lucifearase/mCherry/HSA
HSA/mCherry
• In Depth Sequencing of GenX Lich confirmed that all components of the vector including LTRs , CMV promoter, Triple Fusion Protein, WPRE are present and of correct sequence
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Comparing the proteins expression aWer transduc0on with Gen2 and GenX Lich VSVg virus in 293T cells
• Use various amounts of Gen2 and GenX Lich DNA to make virus (1-‐6μg of DNA)
• Transduce 293 T cells with Gen2 and GenX VSVg virus
• Monitor expression of Luciferase and mCherry proteins under the same experimental condi0ons
LTR CMV mCherry WPRE LTR HSA Luciferase
LTR CMV mCherry WPRE LTR IRES Luciferase Gen2 Lich:
GenX Lich:
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Gen2 Lich (4μg) Transduced 293Ts
mCherry (direct fluorescence) Luciferase:Zenon 647 DAPI
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GenX Lich (4μg) Transduced 293Ts
mCherry (direct fluorescence) HSA:FITC DAPI
• HSA and mCherry were co-‐expressed in all cells examined
• No Luciferase expression was detected in these cells
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mCherry (direct fluorescence) Luciferase:Zenon 647 DAPI GenX Lich (4μg) Transduced 293Ts
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Genera0on of Lich GenY
• Generate vectors expressing other fluorescence proteins (e.g. mCherry instead of iRFP670)
• Infect animals with both viruses and study: – how viral envelopes determine target cell tropism (rectal transmission and other projects)
– the role of VPX in target cells preference (other projects) – perform 0me course experiments (other projects)
LTR CMV iRFP670 WPRE LTR HSA Luciferase IRES
LTR CMV mCherry WPRE LTR HSA Luciferase IRES
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State of the Project • Rectal biopsies are crucial for iden0fica0on of infected cell foci by IVIS • IVIS can direct us to the regions within the 0ssue that contains the
infected cells • Infected cells in the rectum were confirmed by an0body staining and
spectral imaging; nested PCR is in works • Phenotyping of infected cells in the 0ssue is currently in progress • We have op0mized protocol for isola0on, freezing down and infec0on
of primary cells isolated from rectal 0ssue • Luciferase gene cloned between CMV and IRES is op0mal for
Luciferase expression while HSA in front of the mCherry increases expression of mCherry and renders it membranous
• Cloning of mCherry and iRFP670 variants that have such arrangement of Luciferase and fluorescent protein is underway