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Related Experiment Videos

Somatic cell hybrid selection with a transfectable dominant marker.

F C Riera, S B Blam, N N Teng

    Somatic Cell and Molecular Genetics
    |March 1, 1984
    PubMed
    Summary

    The pSV2-neo vector offers an efficient method for selecting G-418 resistant cells, outperforming classical techniques. This dominant marker proved effective in somatic cell hybrid selection, comparable to ouabain, and facilitated the creation of effective fusion partners for human B lymphocytes.

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    Area of Science:

    • Molecular Biology
    • Immunology
    • Cell Biology

    Background:

    • The selection of specific cell populations is crucial in various biological research applications.
    • Traditional methods for selecting mutant cells can be time-consuming and less efficient.
    • Dominant selection markers offer a promising alternative for streamlining cell selection processes.

    Purpose of the Study:

    • To evaluate the efficiency of the pSV2-neo vector as a dominant selection marker in mammalian cell transfection and hybrid selection.
    • To compare the efficacy of pSV2-neo with existing selection markers like ouabain.
    • To assess the utility of pSV2-neo in generating hybrid cell lines for further research, particularly with human B lymphocytes.

    Main Methods:

    • Transfection of mouse myeloma cells (X63-Ag8.653) with the pSV2-neo vector using a modified protoplast fusion technique.

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  • Selection of G-418 resistant cells and determination of transfection efficiency.
  • Fusion of pSV2-neo transfected cells with human myeloma cells (U-266) and selection of hybrids using HAT medium combined with G-418 or ouabain.
  • Reciprocal fusion experiments involving HAT-sensitive, mutant U-266 cells transfected with pSV2-neo.

    • Main Results:

    • The pSV2-neo vector enabled the rapid selection of G-418 resistant cells with an efficiency of 10(-4)-10(-5) within 20 days.
    • pSV2-neo demonstrated comparable efficiency to ouabain in selecting viable hybrid colonies and maintaining immunoglobulin secretion levels.
    • HAT-sensitive hybrid heteromyelomas were successfully generated, proving effective for fusion with human B lymphocytes.

    Conclusions:

    • The pSV2-neo vector serves as an efficient dominant selection marker, significantly improving upon classical cell selection methods.
    • This vector is a valuable tool for somatic cell hybrid selection, offering advantages in efficiency and hybrid quality.
    • The use of pSV2-neo facilitates the generation of versatile hybrid cell lines, including those suitable for fusion with primary human B lymphocytes.