Abstract
To further analyze the VDJ recombination defect in lymphoid pre-B cells from mice with severe combined immune deficiency (scid mice), we have assayed the ability of Abelson murine leukemia virus (A-MuLV) transformed pre-B cells from scid mice to rearrange a recombination substrate in which inverted VH to DJH joins activate a selectable (gpt) gene. In unselected populations, substrate rearrangements occurred frequently, but were aberrant and probably analogous to the aberrant rearrangements observed at endogenous scid Ig gene loci. In contrast, populations of scid pre-B lines selected for gpt activity within the substrate contained mostly "normal" VH to DJH joins within the introduced substrate. These findings demonstrate that scid pre-B cells can make normal joins at low efficiency and are discussed with respect to the potential mechanism of the scid defect and the occurrence of Igs in leaky scid mice.
Highlights
MethodsDerivation, cell culture, and characteristics of A-MuLVtransformed normal 38B9 and 300-18P cells, and scid SC7, SC24, and SC44 cell lines have been previously described (1, 9, 16)
These findings demonstrate that scid pre-B cells can make normal joins at low efficiency and are discussed with re
Summary
Derivation, cell culture, and characteristics of A-MuLVtransformed normal 38B9 and 300-18P cells, and scid SC7, SC24, and SC44 cell lines have been previously described (1, 9, 16). Isolation of neo- and gpt-expressing cell populations and subcloning by limiting dilution were as described (17). Construction of the RV DJH recombination substrate, production of retrovirus, lymphocyte infection, analysis of the integrated RVDJH construct by Southern blotting, and characteristics of the pM7 probe have been previously described (17). Transfections of the pJH195 recombination substrate into A-MuLV cell lines, rescue of the introduced plasmid DNA, transformation of DH5 bacteria and selection on ampicillin and ampicillin + chloramphenicol-supplemented media were as described (13, 18)
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