Ask the IBC Experts Use of Recombinant DNA in Laboratory Animals

David Samols, Ph.D

Nanette R. Kleinman, DVM

Associate Director

Animal Resource Center

Rolf Renne, Ph.D

IBC Contact Information

IBC AdministratorNarinder K. Dhaliwalnkd3@po.cwru.edu

368-6993

IBC AssistantMaureen Dore- Arshenovitz

mxd4@po.cwru.edu368-6925

• IBC ChairDr. David Samolsdrs@po.cwru.edu

368-3520

CWRU IBC Email IBC@resadmin.ra.cwru.edu

• IBC Website http://ora.ra.cwru.edu/main_institutional_biosafety_committee_page.htm

IBC Membership

No fewer than five members:• A Biological Safety Officer • One individual with expertise in plant, plant

pathogen, or plant pest containment principles

• One individual with expertise in animal containment principles

• At least two members shall not be affiliated with the institution

IBC Oversight

Types of IBC Oversight/Review:• Institutional Biosafety Committee approval, review

by the Recombinant DNA Advisory Committee (RAC) that advises the Secretary of HHS, and NIH Approval before initiation.

• NIH and Institutional Biosafety Committee approval before initiation.

• Institutional Biosafety Committee approval before initiation.

• Institutional Biosafety Committee notice simultaneous with initiation

• Exempt Experiments

IBC Approval Before Initiation

• Experiments using human or animal pathogens (Class 2, Class 3, Class 4, or Class 5 Agents as host-vector systems.

• Experiments involving the use of infectious animal or plant DNA or RNA

• Viruses or defective animal or plant DNA or RNA viruses in the presence of helper virus in tissue culture systems.

• Experiments involving whole animals.– Production of BL2, BL3, or BL4 transgenic animals– Gene therapy studies in animals

• Experiments involving whole plants.• Experiments involving more than 10 liters of culture.

IBC Notification Simultaneous with Initiation

• Experiments involving the formation of recombinant DNA molecules containing no more than than two-thirds of the genome of any eukaryotic virus.

• Experiments involving whole plants (not covered in III-C.4).• Section III-E-3. Experiments Involving Transgenic Rodents

– This section covers experiments involving the generation of rodents in which the animal's genome has been altered by stable introduction of recombinant DNA, or DNA derived therefrom, into the germ-line (transgenic rodents). Only experiments that require BL1 containment are covered under this section; experiments that require BL2, BL3, or BL4 containment are covered under Section III-D-4, Experiments Involving Whole Animals.

Exempt Procedures

• Section III-F-1. Those that are not in organisms or viruses.• Section III-F-2. Those that consist entirely of DNA segments from a single

nonchromosomal or viral DNA source • Section III-F-3. Those that consist entirely of DNA from a prokaryotic host

including its indigenous plasmids or viruses when propagated only in that host), or when transferred to another host by well established physiological means.

• Section III-F-4. Those that consist entirely of DNA from an eukaryotic host including its chloroplasts, mitochondria, or plasmids (but excluding viruses) when propagated only in that host  

• Section III-F-5. Those that consist entirely of DNA segments from different species that exchange DNA by known physiological processes

• Section III-F-6. Those that do not present a significant risk to health or the environment as determined by the NIH Director, with the advice of the RAC, and following appropriate notice and opportunity for public comment.

Experiments Involving Whole Animals

• Experiments involving viable recombinant DNA-modified microorganisms tested on whole animals

• For the latter, other than viruses which are only vertically transmitted– Biosafety Level 2

minimum

• Gene therapy studies– Biosafety Level 2

minimum

Section III-D-4. • Covers experiments involving whole

animals • Transgenic animals

– the animal's genome has been altered by stable introduction of recombinant DNA, or DNA derived therefrom, into the germ-line)

– Biosafety Level 1

Purchase/Transfer of Transgenic Rodents

Section III-D-4-c-(2)• The purchase or

transfer of transgenic rodents is exempt from the NIH Guidelines

Identification of Animals

Appendix Q-II-A-1-b-(1)• All genetically engineered

neonates shall be permanently marked within 72 hours after birth, if their size permits.

• If their size does not permit marking, their containers should be marked.

• Transgenic animals should contain distinct and biochemically assayable DNA sequences that allow identification of transgenic animals from among non-transgenic animals.

Disposal of Animals (BL1-N through BL4-N)

Appendix Q-I-B-1. • When an animal containing recombinant DNA or a

recombinant DNA-derived organism is euthanized or dies, the carcass shall be disposed of to avoid its use as food for human beings or animals unless food use is specifically authorized by an appropriate Federal agency.

Appendix Q-I-B-2. • A permanent record shall be maintained of the

experimental use and disposal of each animal or group of animals

Containment

• Animal Biosafety Level 1- ABSL1

• Animal Biosafety Level 2 – ABSL2

• Animal Biosafety Level 3 – ABSL3

• Animal Biosafety Level 4 – ABSL4

Biosafety Level 1 (BL1)• Limited access to lab• Frequent decontamination of surfaces• Autoclaving of waste• Mechanical pipetting; minimize aerosols• No food• Hand washing• No feral rodents, insects• At least Biosafety Level 1 (BL1) lab practices should

be used with all recombinant DNA work, even if the work is exempt from NIH Guidelines.

Biosafety Level 2 (BL2)

• All BL1 requirements, plus:

• Waste is decontaminated off-site from lab

• Biosafety signs on door

• Protective clothing required

• Accidents and exposure must be reported

• May have serum testing for lab staff

Biosafety Level 3 (BL3)

• All BL1 and BL2 requirements, plus:

• Restricted access and biohazard signs

• Rigorous decontamination

• Facilities: biological safety cabinets, HEPA filters, sinks, doors, airlocks, etc.

• Protective activities: clothing, insect control, animal handling, autoclaving, other

Biosafety Level 4 (BL4)

• All BL1-BL3 requirements, plus:• Special decontamination, dunk tanks, fumigation chambers,

double doors, airlocks• High security access, logbook, clothing changes, shower

required• Special accident reporting• High containment animal cages• One-piece positive pressure life-support suits available• Maximum containment facility, double-doored autoclaves,

airflow requirements and monitors

In practice, combinations of containment devices and

procedures may apply and these should be evaluated by the IBC

and other responsible parties

CWRU Animal Biosafety Levels

• Animal Biosafety Level 1- ABSL1– Available for all species

• Animal Biosafety Level 2 – ABSL2– Available for rodents– By special arrangement for other species

• Animal Biosafety Level 3 – ABSL3– Available for infectious prion containing rodents only

• Animal Biosafety Level 4 – ABSL4– Not available

Recombinant Viral VectorsAdenovirus: • Biosafety Level 2 (BL2) Adeno-associated virus: • In the absence of a helper virus" can in

most cases be handled at Biosafety Level 1

Herpesvirus: • Biosafety Level 2 (BL2). Lentivirus: • lentiviruses that are incapable of

establishing productive infections in humans (FIV, EIAV) be conducted at Biosafety Level 2 (BL2).

• Simian or human lentiviruses (SIV, HIV) is typically conducted at a higher containment level.

Poxvirus:

• Biosafety Level 2 (BL2)

Retrovirus:

• Biosafety level 2 (BL2), amphotropic and xenotropic strains of murine leukemia virus

• Biosafety Level 1 , ecotropic vectors with no ability to infect human cells,

Baculovirus:

• Exempt from these registration requirements

Adenovirus

• Infectious human viruses causing mild respiratory disease,

• Biosafety Level 2• Particular care should be given to vectors

containing genes that make products that may be similar to products made by the deleted adenovirus genes.

Adeno-associated virus

• Infectious human viruses with no known disease association.

• Biosafety Level 1– For (AAV) types 1 through 4, and recombinant AAV

constructs, in which the transgene does not encode either a potentially tumorigenic gene product or a toxin molecule and are produced in the absence of a helper virus"

Herpesvirus

• Include infectious human viruses, can cause encephalitis in rare cases

• Herpes simplex virus type-1 (HSV-1), most commonly used.

• Ability to transduce neurons, and large insert capacity.

• Biosafety Level 2

Lentivirus

• Subset of retroviruses, • Infect non-dividing cells• Can cause severe immunologic and neurologic disease in

their natural hosts. • Biosafety Level 2

– For work with non-human lentiviruses that are incapable of establishing productive infections in humans (FIV, EIAV) be conducted at

• Consult the IBC– For work with simian or human lentiviruses (SIV, HIV) is

typically conducted at a higher containment level

Poxvirus

• Include avian viruses (avipox vectors) such as NYVAC and ALVAC, which cannot establish productive infections in humans, as well as mammalian poxviruses which can productively infect humans -- such as vaccinia virus and modified vaccinia viruses (MVA).

• Vaccinia virus can (rarely) cause severe infections in immunocompromised persons, persons with certain underlying skin conditions, or pregnant women

• Biosafety Level 2

Retrovirus

• Can integrate into transduced cells with high frequency

• May have oncogenic potential

• Ecotropic viruses (which can infect only murine cells)

• Amphotropic viruses (which can infect human cells)

• Pseudotyped viruses (which can also infect human cells

• Biosafety Level 2 – For amphotropic and xenotropic strains of murine leukemia virus

• Biosafety Level 1 – For ecotropic vectors with no ability to infect human cells,

Baculovirus

• Non-mammalian virus vectors • Exempt from these registration requirements.