Transfection of human lymphocytes with cloned Epstein-Barr virus (EBV) DNA.
Human primary cord-blood lymphocytes were transfected, using the DEAE-dextran technique, with a set of seven largely overlapping clones jointly covering the whole M-ABA Epstein-Barr virus (EBV) genome. Three fragments, cosmids cMB-14 and cM301-99 and plasmid pM966-20, were able to stimulate transient cellular DNA synthesis, blastic transformation, and clumps formation, as well as to prolong the life span from a maximum of 2 weeks in control cultures to up to 6 weeks. The fragments stimulating DNA synthesis also expressed this property in mutual combinations or when combined with cosmid cMSal-A or cM302-21. Their use with any other fragments in cotransfection did not result in further DNA synthesis stimulation. Cosmids cM302-23 and cMSal-B suppressed this effect. Cosmid cM301-99 but not cM302-23 induced transient EBNA-1 formation in about 1% of lymphocytes. Lymphocytes transfected with single fragments or their combinations failed to grow into immortalized cell lines. The results suggest that transient expression of viral functions at levels achievable by transfection is not sufficient for cell immortalization.
D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000235:genetics*; D005260:Female; D005312:Fetal Blood; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D008214:Lymphocytes / Q000382:microbiology*; D011247:Pregnancy; D014162:Transfection
D004279:DNA, Viral; D004262:DNA Restriction Enzymes