22 Expression in Eukaryotic Cells: Gene Transfer Using Retroviral Vectors
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requirements of the target cells. The G418 concentration necessary to select
the target cells should be determined in advance and may be different from
the 400 Jlg/ml used for selection of ecotropic and amphotropic packaging
cells. A G418 concentration that kills the non-resistant target cells slowly
over a period of 5-8 days should be used.
Results
Assessment of transfection efficacy
The presence of the protein encoded by the cDNA inserted into the genome
of the target cell can be easily detected by Western blotting or immunoprecipitation (see Chapter 24), if a suitable antibody has been prepared or if one
is commercially available. Expression levels in different target cells or using
different transfection strategies may be compared in celllysates after adjusting for total protein content. The cellular localization of the protein can
be investigated by immunocytochemistry. If the protein expressed by the
target cells is secreted into the medium, its presence in the supernatants can
be investigated by ELISA, Western blotting or immunoprecipitation. If the
protein of interest exhibits enzymatic activity, a substrate assay can be used
instead or in combination with the immunological methods.
Comments
Biohazard issues
The specific biosafety issues raised when working with retroviral vectors
should be discussed in advance for any given protocol with the local biosafety comittee. In several countries, strict governmental standards or laws
have been set up which need tobe considered carefully. The review of experimental protocols by governmental institutions may require several
months. These time requirements need to be considered when planning
the experimental work.
The ecotropic viruses cannot replicate in non-rodent cells. Release of
replication-competent helper viruses has never been reported with the
second generation ecotropic packaging cell lines \j/CRIP CRE (15) and
GP+E86 (16), whereas the use of\j/2 cells (13) carries a small but well known
risk. The amphotropic viruses can bind to human cells and can integrate the
provirus into the human genome. However, due to other levels ofhost range
349
requirements of the target cells. The G418 concentration necessary to select
the target cells should be determined in advance and may be different from
the 400 Jlg/ml used for selection of ecotropic and amphotropic packaging
cells. A G418 concentration that kills the non-resistant target cells slowly
over a period of 5-8 days should be used.
Results
Assessment of transfection efficacy
The presence of the protein encoded by the cDNA inserted into the genome
of the target cell can be easily detected by Western blotting or immunoprecipitation (see Chapter 24), if a suitable antibody has been prepared or if one
is commercially available. Expression levels in different target cells or using
different transfection strategies may be compared in celllysates after adjusting for total protein content. The cellular localization of the protein can
be investigated by immunocytochemistry. If the protein expressed by the
target cells is secreted into the medium, its presence in the supernatants can
be investigated by ELISA, Western blotting or immunoprecipitation. If the
protein of interest exhibits enzymatic activity, a substrate assay can be used
instead or in combination with the immunological methods.
Comments
Biohazard issues
The specific biosafety issues raised when working with retroviral vectors
should be discussed in advance for any given protocol with the local biosafety comittee. In several countries, strict governmental standards or laws
have been set up which need tobe considered carefully. The review of experimental protocols by governmental institutions may require several
months. These time requirements need to be considered when planning
the experimental work.
The ecotropic viruses cannot replicate in non-rodent cells. Release of
replication-competent helper viruses has never been reported with the
second generation ecotropic packaging cell lines \j/CRIP CRE (15) and
GP+E86 (16), whereas the use of\j/2 cells (13) carries a small but well known
risk. The amphotropic viruses can bind to human cells and can integrate the
provirus into the human genome. However, due to other levels ofhost range