A method for infection of cultured myogenic cells with rous sarcoma virus using polybrene

L. M. Sorokin, E. H. Morgan, G. C.T. Yeoh, R. Coelen

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    Abstract

    A method for efficiently infecting primary myogenic cultures with a temperature-sensitive variant of the Prague strain of Rous sarcoma virus (RSV, tsLA24) to obtain a high yield of transformed myogenic cells is reported. It incorporates the use of an amorphous polymer of polycations, Polybrene, to enhance the absorption of the virus by the muscle cells. In addition, other steps which were shown to be important were a) to allow cell attachment before infection, b) to infect at 35° C in low protein medium, c) to use a density of 1 to 1.5×106 cells/60-mm dish, d) gentle agitation during infection, and e) to minimize the number of passages after infection. The use of the temperature-sensitive virus provided a means of confirming the presence of myogenic cells in transformed cultures. When infected cells were maintained at 35° C (the permissive temperature for virus activity) they exhibited the characteristics of transformed cells. These characteristics included altered cell morphology, the absence of contact-inhibited growth, growth in semisolid medium, and expression of the src oncogene. In contrast, not express src and showed normal myogenic development and ultimately formed myotubes.

    Original languageEnglish
    Pages (from-to)63-68
    Number of pages6
    JournalIn Vitro Cellular & Developmental Biology
    Volume25
    Issue number1
    DOIs
    Publication statusPublished - 1 Jan 1989

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