350
MARTIN MARX
restriction the helper virus probably cannot replicate in human cells. To
date, replication in humans has not been reported. Both ecotropic and amphotropic viruses must be treated as potential biohazards. The minimal requirements when working with all the viruses include the exclusive use of
laminar flow hoods, protective gloves and clothing, disinfection of all utensils under the laminar flow hood, disposal in a biohazard bag and sterilization of all waste in an autoclave.
The transfected target cells must be assayed for the presence of replication-competent helper virus. Several radioactive and non-radioactive assays for the detection of retroviral reverse transcriptase activity are commercially available. Alternatively, helper virus can be detected through horizontal spread of drug resistance. In this sensitive assay the supernatants of
the transfected target cells are assayed for the presence of virus carrying the
selectable marker neomycin resistance. The most suitable assay for a given
experimental protocol should be discussed with the local biosafety comittee
or with an experienced retrovirologist.
References
1. Weiss K, Teich N, Varmus H, Coffin J (Eds.), (1985) Retroviruses as Vectors ofForeign Genes, in Molecular Biology of Tumor Viruses, 2nd Edition, pp. 36-73, Cold
Spring Harbor Laboratory 1985, New York
2. Jorgensen RA, Rothstein SJ, ReznikoffWS (1979) A Restrietion Enzyme Cleavage
Map of Tn5 and Location of a Region Encoding Neomycin Resistance. Mol Gen
Genet 177:65-72
3. Miller AD, Rosman GJ (1989) Improved Retroviral Vectors for Gene Transfer and
Expression. BioTechniques 7:980-990
4. Rosenberg SA, Aebersold P, Cornetta K, Kasid A, Morgan RA, Moen R, Karson EM,
Lotze MT, Yang JC, Topalian SL, Merino MJ, Culver K, Miller AD, Blaese RM, Anderson WF (1990) Gene Transfer into Humans - Immunotherapy of Patients with
Advanced Melanoma, Using Tumor-Infiltrating Lymphocytes Modified by Retroviral Gene Transduction. N Engl J Med 323:570-578
5. Veillette A, Fournel M ( 1990) The CD4 Associated Tyrosine Protein Kinase p56lck Is
Positively Regulated through its Site of Autophosphorylation. Oncogene 5:14551462
6. Lynch CM, Clowes MM, Osborne WRA, Clowes AW, MillerAD (1992) Long-Term
Expression ofHuman Adenosine Deaminase in Vascular Smooth Museie Cells of
Rats: A Model for Gene Therapy. Proc Natl Acad Sei USA 89:1138-1142
7. Nolta JA, Xiao JY, Bahner I, Kohn DB (1992) Retroviral-Mediated Transfer of the
Human Glucocerebrosidase Gene into Cultured Gaueher Bone Marrow. J Clin Invest 90:342-348
8. Haughn L, Gratton S, Caron L, SekalyRP, Veillette A, Julius M (1992) Association of
Tyrosine Kinase p56lck with CD4 Inhibits the Induction ofGrowththrough the aß
T-Cell Receptor. Nature 358:328-331
MARTIN MARX
restriction the helper virus probably cannot replicate in human cells. To
date, replication in humans has not been reported. Both ecotropic and amphotropic viruses must be treated as potential biohazards. The minimal requirements when working with all the viruses include the exclusive use of
laminar flow hoods, protective gloves and clothing, disinfection of all utensils under the laminar flow hood, disposal in a biohazard bag and sterilization of all waste in an autoclave.
The transfected target cells must be assayed for the presence of replication-competent helper virus. Several radioactive and non-radioactive assays for the detection of retroviral reverse transcriptase activity are commercially available. Alternatively, helper virus can be detected through horizontal spread of drug resistance. In this sensitive assay the supernatants of
the transfected target cells are assayed for the presence of virus carrying the
selectable marker neomycin resistance. The most suitable assay for a given
experimental protocol should be discussed with the local biosafety comittee
or with an experienced retrovirologist.
References
1. Weiss K, Teich N, Varmus H, Coffin J (Eds.), (1985) Retroviruses as Vectors ofForeign Genes, in Molecular Biology of Tumor Viruses, 2nd Edition, pp. 36-73, Cold
Spring Harbor Laboratory 1985, New York
2. Jorgensen RA, Rothstein SJ, ReznikoffWS (1979) A Restrietion Enzyme Cleavage
Map of Tn5 and Location of a Region Encoding Neomycin Resistance. Mol Gen
Genet 177:65-72
3. Miller AD, Rosman GJ (1989) Improved Retroviral Vectors for Gene Transfer and
Expression. BioTechniques 7:980-990
4. Rosenberg SA, Aebersold P, Cornetta K, Kasid A, Morgan RA, Moen R, Karson EM,
Lotze MT, Yang JC, Topalian SL, Merino MJ, Culver K, Miller AD, Blaese RM, Anderson WF (1990) Gene Transfer into Humans - Immunotherapy of Patients with
Advanced Melanoma, Using Tumor-Infiltrating Lymphocytes Modified by Retroviral Gene Transduction. N Engl J Med 323:570-578
5. Veillette A, Fournel M ( 1990) The CD4 Associated Tyrosine Protein Kinase p56lck Is
Positively Regulated through its Site of Autophosphorylation. Oncogene 5:14551462
6. Lynch CM, Clowes MM, Osborne WRA, Clowes AW, MillerAD (1992) Long-Term
Expression ofHuman Adenosine Deaminase in Vascular Smooth Museie Cells of
Rats: A Model for Gene Therapy. Proc Natl Acad Sei USA 89:1138-1142
7. Nolta JA, Xiao JY, Bahner I, Kohn DB (1992) Retroviral-Mediated Transfer of the
Human Glucocerebrosidase Gene into Cultured Gaueher Bone Marrow. J Clin Invest 90:342-348
8. Haughn L, Gratton S, Caron L, SekalyRP, Veillette A, Julius M (1992) Association of
Tyrosine Kinase p56lck with CD4 Inhibits the Induction ofGrowththrough the aß
T-Cell Receptor. Nature 358:328-331