the cfar biohazard animal core
DESCRIPTION
The CFAR Biohazard Animal Core. Director: John Chan, MD Co-Director: Larry Herbst, PhD Staff: Jiayong Xu , BA Location:AECOM, Chanin Bldg AECOM, Price Bldg. The Goal of the Biohazard Animal Core. - PowerPoint PPT PresentationTRANSCRIPT
The CFAR Biohazard Animal Core
Director: John Chan, MDCo-Director: Larry Herbst, PhD
Staff: Jiayong Xu, BA
Location: AECOM, Chanin BldgAECOM, Price Bldg
The Goal of the Biohazard Animal Core
To provide a biosafety level 3 (BSL 3) containment facility for studies involving in vivo modeling of infection with HIV-1 and AIDS-associated pathogens using various murine experimental models
Implementation of the Goals of the Biohazard Animal Core
To provide a safe laboratory environment for the performance of experiments involving biohazardous pathogens that require a BSL 3 containment facility.
To provide reagents, including specific strains of pathogens, training, and technical assistance to facilitate research using murine infectious disease models involving HIV-1 and AIDS-associated pathogens such as Mycobacterium tuberculosis and Cryptococcus neoformans.
To provide comprehensive services to researchers not familiar with studies involving infection of mouse with biohazardous pathogens, which are designed to investigate pathogen-host interaction, pathogen-pathogen interaction, drug discovery, as well as mechanisms involved in pathogenesis and host defense.
To provide assistance in the study design and data interpretation of in vivo studies involving the use of murine models of infection.
To provide support in breeding, genotyping, and maintenance of specific transgenic and gene-knockout mouse strains.
• The Chanin lab (~1,500 sq ft): 2,000 mice
• The Price lab (~2,000 sq ft): 2,200 mice
Capacity of the Animal Core
Infection by Aerogenic Challenge
We have extensive experience in infecting mice with Mycobacterium tuberculosis via aerosols using the "Wisconsin" chamber or the In-Tox "nose-only" aerosolization apparatus and will optimize conditions for the aerosolization of other pathogens
The Chanin Aerosolization Machine
The Intox Nose-Only Aerosolization Apparatus
Tis
sue
Bac
teria
l Bur
den
Time
Adaptive Immunity
Initiation of Immunosuppressive
Therapy
Chronic Phase
Reactivation Phase
Low-Dose Reactivation Model of Tuberculosis
Tis
sue
Bac
teri
al B
urd
en
Time
Adaptive Immunity
Initiation of Anti-TB Therapy
Apparent Sterile State
Reactivation Phase
The Cornell Model of Reactivation Tuberculosis
Lung: 3 months Post-Mtb Infection (Paraffin-embedded Tissues
Analysis of Tissues of Pathogen-infected Animals
Lungs: 6 months Post-Mtb Infection (Frozen Sections)
Liver: 6 months Post-Mtb Infection (Laser Capture Microdissection)
Immunofluorescence Staining of Tuberculous Tissues
Immunophenotyping of Cells from Infected Tissues
The SCID-hu Mouse Model
The thy/liv-SCID-hu mice are generated by the core and can be infected with various titered R5, X4, and X4R5 primary isolates of HIV obtained from the BSL3/Virology Core to examine anti-HIV effects of novel therapeutics developed by
CFAR investigators
Thy-liv-SCID-hu mice: An in vivo Model for Evaluating Anti-HIV-1 Therapy
Constructed by implanting human fetal thymus and liver under the kidney capsule of SCID mice.
The peripheral blood and lymphoid tissues of the mice are populated with > 5% human T cells and monocytes.
After i.p. inoculation of HIV-1, human cells in the peritoneum become infected, migrate to lymph nodes and traffic to and infect the human thymic implant.
Mice with Infection of Thymic Implants of thy/liv-SCID-hu HIV-1
Inject HIV-1 into thy/liv SCID-hu mouse
Limiting Co-culture of implant to analyze for HIV infection
Specialty Mouse Strains
Provide a number of mouse strains including SCID, Rag-/-, Rag2-/-c-/-,
and the NOD (nonobese-diabetic)/SCID/IL2Rnull mouse models.
Cryopreservation of mouse strains
A Novel Transgenic Mouse Model Construct #1: human CD4-P2A-CCR5
E: mCD4 Enhancer P: mCD4 Promotor
2
4
3 SalI SalI
hCD4 hCCR5P2A1
Primer 1: ACGC GTCGACGCCACCATGAACCGGGGAGTCCCTTTTAG
Primer 2: CTGCTTGCTTTAACAGAGAGAAGTTCGTGGC TCCGGAACCAATGGGGCTACATGTCTTC
Primer 3: GCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCC atggattatcaagtgtcaag
Primer 4: TTCCGCGGCCGCTATGGCCGAC GTCGACTCACAAGCCCACAGATATTTC
SalI CD4
P2A CD4
P2A CCR5
CCR5SalI
Construct # 2: human Cyclin T1
10 1 10 2 10 3 10 4 10 5
10 1
10 2
10 3
10 4
10 5
10 1 10 2 10 3 10 4 10 5
10 1
10 2
10 3
10 4
10 5
101 102 103 104 105
101
102
103
104
1050 0
0.9499.1
F1 progeny transgenic mouse
3.3513.7
10 1 10 2 10 3 10 4 10 5
10 1
10 2
10 3
10 4
10 510.3 1.16
4.3884.1
10 1 10 2 10 3 10 4 10 5
10 1
10 2
10 3
10 4
10 510.6 0.93
2.6885.7
10 1 10 2 10 3 10 4 10 5
10 1
10 2
10 3
10 4
10 5 53% 30%
4.7911.7
12.9 0.86
4.381.9
10 1 10 2 10 3 10 4 10 5
10 1
10 2
10 3
10 4
10 5 72% 10%
1.8215.9
101 102 10 3 104 10 5
101
102
103
104
1053.65 0
0.7395.6
Control mouse No. 724 No. 726 No. 730
Mouse CD4 cells
Mouse CD8 cells
Human CCR5
Hu
man
CD
4
Selective expression of hCD4 and CCR5 by mouse CD4 cells. PBMCs were isolated from a control mouse and three F1 progeny of founder mouse No.479. Expression of hCD4 and CCR5 by the mouse CD4+ cells and CD8+ cells was analyzed by two-color flow cytometry. The percentage of positive cells in each quadrant is indicated.
A Novel Transgenic HIV Mouse Model
51% 32%
The Gnotobiotic Facility
In vivo Imaging: the IVIS Spectrum
The IVIS
IVIS: Tracking Malaria Infection
CQ: Chloroquine; PQ: Primaquine; Ato: Atovaquone
Behavioral Study of NeuroAIDS
Other Animal Procedures
post-mortem dissection for the procurement of infected organs
retro-orbital bleed
delivery of pharmacological or biological agents by various routes (oral, subcutaneous, intramuscular, intravenous, respiratory, intraperitoneal, retro-orbital and intracranial)
Provide biohazard barrier housing and husbandry for mice infected with HIV and various AIDS-related pathogens
Provide training for experimental techniques involved in conducting animal BSL3 experiments with various pathogens
Accomplishments of the Animal Core• Acquisition of the in vitro imaging system: IVIS Spectrum
• The NeuroAIDS model, including the establishment of the radial water maze for behavorial study (addition of 500 sq ft)
• Establishment of a novel HIV mouse model: mice transgenic for expression of human CD4, CCR5 and Cyclin T1 that display successful in vitro and in vivo HIV infection.
• Novel mouse strains: Rag2-/-c-/-, and the NOD/SCID/IL2Rnull mouse models
• Additional 1,500 sq ft BSL3 facility (2,200 mice)
• New programs: interactions between HIV and M. tuberculosis, Malaria, and Herpes Virus; in vivo analysis of neuroAIDS in mice
• Publication (2008-2011): 36
• Users: the Core supports the research of 25 investigators (2011 progress report; 21 major users); first progress report of 2004 listed 8 investigators as major users
Acknowledgements
• CFAR Cores and Staff: Virology, Developmental, Flow Cytometry, Immunology/Pathology, Clinical Cores.
• CFAR investigators and collaborators• Albert Einstein College of Medicine/Montefiore
Medical Center