Molecular cloning and analysis of lymphokines / edited by David R. Webb, David V. Goeddel.

Date:
1987
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Molecular cloning and analysis of lymphokines / edited by David R. Webb, David V. Goeddel. Source: Wellcome Collection.

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    IDENTIFICATION AND EXPRESSION OF cDNA PLASMIDS 25 tr^Q. Pvul O) CD С CL Fig. 1. Physical and genetic map of the expression vector pSV529. The thin segment part of the circle represents the pBR322 DNA sequence. SV40 sequences are shown as a heavy line and contain a partially duplicated DNA replication and early region of the SV40 genome. The coding regions for the early and late proteins are shown as dark arrows. The dotted 5' ends and wavy poly(A) 3' ends indicate the span of the mRNAs [for more details on the construction of pSV529, refer to Gheysen and Fiers (1982)]. A gene inserted into the unique BamHl site of pSV529 is under transcriptional control of the late SV40 pro- motor. This figure is reproduced with permission from Gheysen and Fiers (1982), Ex¬ pression and excretion of human IFN-ß in monkey cells after transfection with a recombi¬ nant SV40 plasmid vector,/. Mol. Appi. Genet. 1, 385. cloning vector since they suggested that—providing the cDNA copy is (1) full length and (2) inserted in the correct orientation relative to the SV40 late promotor—a sensitive screening method could be derived based on direct expression. Double stranded cDNA was synthesized using sucrose gradient-frac¬ tionated, human spleen-derived IL-2-specifìc and IFN-7-specifíc mRNA from a single donor essentially as described in Devos et al. (1979), frac¬ tionated by Polyacrylamide gel electrophoresis. Appropriate size classes (ranging between 800 and 1000, 1000 and 1250, and 1250 and 1400 bp for IFN-7, and ranging between 600 and 750, and 750 and 1000 bp for IL-2) were inserted into the unique BamWl site of pSV529 DNA (Fig, 1) via