( by varying the multiplicity of infectio...)High-Titer ,Retroviral Vectors for Gene Delivery

by varying the multiplicity of infection, in contrast to standard transfection methods for which typically a small population of transfected cells are capable of the uptake and stable integration of vector DNA and for which the copy number is unpredicatable and often prohibitively high for many applications.³ The combination of high transduction efficiency and copy number control make retroviral delivery systems particularly useful for mammalian cDNA expression library production and screening. In these types of experiments, transduction of a large number of target cells with single-copy cDNA expression cassettes is highly desirable.⁴ The ability to efficiently transduce a wide range of hard-to-transfect cell types has also facilitated the reconstruction of transgenic animals. Additionally, retroviral vectors have become the system of choice for the in vivo delivery of therapeutic genes in the clinic.

We introduce the retroviral vectors pFB and pFB-Neo, which are based on MMLV and contain an extended packaging sequence and splice-site configuration that increases viral titer and gene expression from the viral promoter.⁵ These vectors can be used with any MMLV-based packaging cell line. MMLV-based virions are labile and readily inactivated by ethanol, ultraviolet light, and human complement.⁶ Thus, virus produced with these vectors, using packaging cell lines that allow infection of human cells (e.g., amphotropic packaging cells), are relatively safe and can be used in a P2 tissue culture facility.

We have achieved titers in excess of 10⁸ cfu/ml with pFB and greater than 10⁷ cfu/ml with pFB-Neo using an amphotropic packaging cell line; we found that expression levels for these vectors were generally comparable to or higher than those for other commercially available retroviral vectors.

Replication-Defective Retroviral Gene Tran
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