       Document 0690
 DOCN  M9640690
 TI    Stable transfection of cloned murine T helper cells.
 DT    9604
 AU    Will A; Rollinghoff M; Gessner A; Institut fur Klinische Mikrobiologie
       und Immunologie,; Universitat Erlangen-Nurberg, Erlangen, Germany.
 SO    J Immunol Methods. 1995 Dec 15;188(1):139-46. Unique Identifier :
       AIDSLINE MED/96140789
 AB    Here we describe a protocol for the stable transfection of murine T
       helper (Th) cells and long term culture of the resulting transfectants.
       The electroporation protocol was established for the murine Th2 clone
       L1/1 by testing different parameters determining the electric field
       (capacitance, voltage, single or twin pulse) as well as the activation
       status of the cells. The transfected T cells were genetically altered by
       stable integration of the neomycin resistance gene, encoded in the
       vector pM5neo, into the genome. For selection and long term culture of
       stable transfectants a scheme combining selection with the antibiotic
       neomycin (G-418, Geneticin) and repeated stimulation with antigen
       presenting cells (APC) and antigen was established. This protocol should
       also be applicable to other antigen reactive T cells. The resistance of
       the T cells to neomycin correlated directly with expression of the
       transferred neomycin resistance gene as demonstrated by mRNA analysis.
       Applying periodic reselection with neomycin the transfected Th2 cells
       were found to be stable for more than 18 months in culture and displayed
       an unaltered antigen recognition and lymphokine production pattern as
       compared with the untransfected L1/1 Th2 cells.
 DE    Animal  Antigen-Presenting Cells/IMMUNOLOGY/METABOLISM  Base Sequence
       Cell Line  Clone Cells/IMMUNOLOGY  Electroporation  Female
       Genes/IMMUNOLOGY  Lymphocyte Transformation/GENETICS  Mice  Mice, Inbred
       BALB C  Molecular Sequence Data  Support, Non-U.S. Gov't  Th2
       Cells/IMMUNOLOGY/*METABOLISM  Transfection/GENETICS/*METHODS  JOURNAL
       ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

