Construction of viral-based linear vectors for mammalian cells

• Main Author: Bhattacherjee, Vasker
• Published: University of Edinburgh 1993
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.641596

The linear vector was based upon a pre-existing circular plasmid which contained replication sequences from Epstein-Barr virus, p220.2. p220.2 was modified by the insertion of two telomeric DNA fragments in opposing orientations separated by a selectable "stuffer" fragment (the kanamycin resistance marker). This construct which contained two potential internal telomeres was named p220.2TC. Excision of the stuffer fragment prior to transfection exposed the termini of the telomeric sequences to create the linear vector, p220.2ET which terminated in telomeric DNA sequences. A second linear molecule, p220.2IT, was obtained from p220.2TC as a control by cleaving the plasmid in the ampicillin resistance marker leaving the telomeric DNA fragments in an internal position. 220.2TC- was designed as a negative control for telomere function. p220.2, p220.2TC, p220.2ET and p220.2IT were transfected into human Raji cells by electroporation. Southern blot analysis of the DNA from transfected cells showed that the vectors were not rearranged by this method. Subsequent experiments did not find evidence that p220.2TC replicates in Raji cells as an episome. The Southern blot analysis showed progressive loss of p220.2TC, p220.2ET and p220.2IT from the cells without replication taking place. Additionally, p220.2TC could not be rescued in <I>E.coli</I>. In these same experiments, however, the control plasmid p220.2 failed to replicate as it did in the initial tests on the Raji cells. Despite many attempts using different experimental conditions, replication or retention of p220.2TC was not detected. Elongation of the telomeric sequences on p220.2ET was also not detected.