BOSC 23 cell line高产量慢病毒包装细胞株 BioVector NTCC质粒载体菌种细胞基因保藏中心

BOSC 23 cell line高产量慢病毒包装细胞株

The generation of high-titer, helper-free retroviruses by transient transection has been achieved by using
the highy transfectable 293T cell line into which are stably
introduced constructs that express retroviral packaging functions. The resulting ecotropic virus packing cell line BOSC
23 produces infectious retrovirus at >106 infectious units/ml
of supernatant within 72 hr after CaPO4-mediated btansfection. A stringent assay for replication-competent virus showed
that no helper virus was present. The system can produce high
titers of retroviral vectors expressing genes that are extremely
difficult to propagate at high titer in stable producer lines. This
method should facilitate and extend the use of helper-free
retroviral gene transfer, as weli as be useful for gene therapy.
Present methods for creating high-titer, helper-free retroviral
stocks employ the creation of a stable producer cell line that
expresses the retroviral vector (1). Producer lines are made
by introducing the retroviral vector into a packaging cell line,
which synthesizes all the proteins required for viral assembly. It is then necessary to screen many clones for one clone
that expresses the retroviral vector at high titer, because
virus production by the original pool of transfected clones is
low. Selection takes at least 1 month, and prolonged high
expression of the retroviral mRNA may slow the growth of
the producer line, allowing outgrowth of poorly producing
variants during prolonged cultivation.
To circumvent these problems, a strategy was devised that
uses transient transfection ofthe retroviral packaging cell line
BOSC 23 to produce high-titer, helper-free infectious retroviruses. The BOSC 23 producer cell line (derived from the
Ad5-transformed human embryonic kidney 293 cell line) is
more transfectable than the currently employed packaging
cell lines. By using BOSC 23 packaging cells and optimizing
the CaPO4 transfection procedure, we have obtained retroviral titers in excess of 107 infectious particles per milliliter of
supernatant within 48-72 hr after transfection. The retroviruses produced are helper-free and can infect early hematopoietic progenitors. Moreover, titers in excess of 106 have
routinely been attained by using retroviral vectors expressing
genes that appear toxic to stable producer lines.


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