Recombinant DNA Advisory Committee - 3/3-4/94 
initiated at a new site (the original Principal Investigator is the same for the new site), 
(4) New site/new Principal Investigator -- RAC-approved protocol initiated at a new site 
(the Principal Investigator for the new site is different than the Principal Investigator of 
approved for the original site), (5) "Umbrella" protocols - RAC-approved protocol 
initiated at more than one additional site (Principal Investigator may be the same or 
different than the Principal Investigator approved for the original site), (6) Modification 
not related to gene transfer - a modification to the clinical protocol that is not related to 
the gene transfer portion of the study, and (7) Gene marking protocols -- RAC-approved 
vector constructs or RAC-approved vector constructs with minor modifications/additional 
target cells. 
XI. ADDITION TO APPENDIX D OF THE NIH GUIDELINES REGARDING 
DELIBERATE TRANSFER OF A CHLORAMPHENICOL RESISTANCE GENE TO AN 
AVIRULENT STRAIN OF RICKETTSIA PROWAZEKl /mL POLICASTRO 
Review— Dr. Post 
Dr. Walters called on Dr. Post to present his primary review of the proposal submitted 
by Dr. Paul F. Policastro of the NIH, Rocky Mountain Laboratories, Hamilton, Montana. 
TTiis request was deferred by the RAC at its June 7-8, 1993, meeting until the 
investigator returned to the full RAC with data demonstrating that the construct is safe 
and useful, and that there is a selective advantage of using chloramphenicol resistance 
over other selectable markers. Dr. Post explained that the investigator is requesting 
permission to introduce the chloramphenicol resistance gene into Rickettsia prowazeki. 
Chloramphenicol is one of the two antibiotics of choice to treat the human infection. 
This antibiotic resistant gene will be used as a selectable marker to establish a 
transformation system in eukaryotic host cells. Such studies will be valuable for 
conducting research on this fatally pathogenic organism. The investigator has responded 
to the RAC's initial concerns as follows: (1) the study will be confined to Rickettsia 
prowazeki Strain E, (2) a cloning vector is proposed that includes several safety features, 
and (3) the experiment will be conducted in a BL3 facility. The chloramphenicol 
resistant strain will be used only to develop the transformation conditions; other 
selectable markers will be developed to perform subsequent research. Although the 
proposal is reasonable with regard to the molecular biology aspects. Dr. Post deferred to 
the opinion of infectious disease experts with regard to safety issues. 
Review-Dr. Straus 
Dr. Straus explained that Strain E Rickettsia is of reduced virulence but is not avirulent. 
Although Strain E has been used as a human vaccine, the strain is probably capable of 
reversion to a virulent form after "n" number of ex vivo passages. In Dr. Policastro's 
submission, he states that Strain E has been reported to revert to virulent phenotypes 
after 13 serial passages in the lungs of albino mice and upon brief passage in pigs under 
heavy inoculation. Symptoms of human infection with Strain E have been observed 
following exposure to high titers, i.e., between 1 x 10^ and 1 x 10^ infectious organisms. 
The proposed experiment will require manipulating the organism at the levels of 1 x lO” 
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Recombinant DNA Research, Volume 19 
