Recombinant adeno-associated virus type 2 (rAAV) vecderived cell line. Effective amplification of rAAV vectors tors have recently been used to achieve long-term, high introduced into 293 cells by infection was also demonlevel transduction in vivo. Further development of rAAV strated. Passage of rAAV with d27.1-rc results in up to 200-vectors for clinical use requires significant technological fold amplification of AAV-GFP with each passage after improvements in large-scale vector production. In order to coinfection of the vectors. Efficient, large-scale production facilitate the production of rAAV vectors, a recombinant (Ͼ10 9 cells) of AAV-GFP from a proviral cell line was also herpes simplex virus type I vector (rHSV-1) which does not achieved and these stocks were free of replication-comproduce ICP27, has been engineered to express the AAVpetent AAV. The described rHSV-1 vector provides a 2 rep and cap genes. The optimal dose of this vector, novel, simple and flexible way to introduce the AAV-2 rep d27.1-rc, for AAV production has been determined and and cap genes and helper virus functions required to proresults in a yield of 380 expression units (EU) of AAV-GFP duce high-titer rAAV preparations from any rAAV proviral produced from 293 cells following transfection with AAVconstruct. The efficiency and potential for scalable delivery GFP plasmid DNA. In addition, d27.1-rc was also efficient of d27.1-rc to producer cell cultures should facilitate the at producing rAAV from cell lines that have an integrated production of sufficient quantities of rAAV vectors for clini-AAV-GFP provirus. Up to 480 EU/cell of AAV-GFP could cal application. be produced from the cell line GFP-92, a proviral, 293

We have developed an Escherichia coli plasmid vector for the identification and expression of foreign DNA segments that are open reading frames (ORFs). The 5' end of ompF, an E. coli gene encoding an abundant outer membrane protein, is used to provide a strong, regulated promoter, translation initiation site, and signal sequence for export from the cytoplasm. This sequence is coupled to the lacZ gene of E. coli so that expression of 3-galactosidase requires ompF transcription and translation signals. However, this hybrid gene is LacZ-because lacZ is out of frame with respect to ompF. Restriction enzyme recognition sites are located between ompF and lacZ to allow convenient insertion of DNA fragments. If an insert is an ORF of the correct length, ompF and lacZ become realigned in frame, resulting in a LacZ' gene that produces a tribrid protein with the translation product of the insert sandwiched between OmpF and J3-galactosidase. The LacZ+ phenotype thus identifies clones containing an expressed ORF. To demonstrate the vector's utility we inserted a fragment from the herpes virus thymidine kinase gene and used the resulting tribrid protein to raise antibodies that precipitate thymidine kinase from herpes virus-infected cells. We also inserted a fragment from the E. coli lexA gene to produce a tribrid protein that is precipitated by antiserum raised with LexA protein. Thus, tribrid fusion proteins can be used to produce or detect antibodies and also to identify the product of a cloned gene.One of the goals of genetic engineering is the expression of all or part of a gene to produce an antigenic protein segment that can be used as a vaccine or to raise or detect antibodies for research and diagnosis. Expressing A(argF-lac)U169 rpsL150 (strR) relAl flbB5301 deoCl ptsF25 malPQ: :Tn5 ompBcsl. The ompR101 mutation (2) drastically decreases ompF expression, whereas the ompBcsl mutation decreases ompF expression at temperatures near 30°C and shows increased ompF expression above 37°C (unpublished data). MH3000 was used to construct LacZ+ clones because it is highly transformable and prevents the overproduction lethality of ompF-lacZ fusions (see Results and Discussion). We also observed that ompR+ cells containing the pORF vectors formed light blue colonies on 5-bromo-4-chloro-3-indolyl-f3-D-galactoside (XG) plates, even though ompF and lacZ were out of frame, whereas with MH3000 the colonies were white, allowing clones to be more clearly identified. TK1046 transformed poorly and was not suitable for the initial construction of clones; its use was therefore limited to testing for overproduction lethality and producing high levels of tribrid proteins. The cs mutation in TK1046 is either in ompR or envZ and allows more expression of ompF at 30°C than does ompR101. Vero The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.