To overcome these obstacles ourlaboratory created new packaging lines, termed Phoenix, and made several improvements to them over the first generation approach (15). Two cell lines were created, Phoenix-ECO and Phoenix-AMPHO. Additionally, a line termed Phoenix-gp has been produced that expresses only gag-pol.

This line is available for further pseudotyping of retroviral virions with other envelope proteins such as gibbon ape leukemia virus envelope or Vesicular Stomatitus VSV-G protein, xenotropic, or retargeting envelopes can also be added.

Both Phoenix-ECO and Phoenix-AMPHO were tested for helper virus production and es tablished as being helper-virus fre and are fully compatible with transient, episomal stable, and library generation for retroviral gene transfer experiments (15):

All lines are capable of carrying episomes for long-term stable production of retrovirus (see below). All lines are readily testable by flow cytometry for stability of gag-pol (CD8) and envelope expression; (after several months of testing the lines appear stable, and do not demonstrate loss of titre as did the first-generation lines BOSC23 and Bing. The new lines are more stable we think partly due to the choice of promoters driving expression of gag-pol and envelope).Both lines can also be used to transiently produce virus in a few days.Thus we have the means to deliver large libraries of retroviruses into nearly any mammalian cell type, mouse or human.

Finally, we have tested the titres produced by these lines. Using standard polybrene-enhanced retroviral infection we observe titres approaching or above 10^7 per ml for both Phoenix-eco and Phoenix-ampho when carrying episo mal constructs (see below). When transiently produced virus is made, titres are usually 1/2 to 1/3 that value.