Figure 2.

Construction of recombinant IgH loci bearing Ter3 and Ter462* mutations. Illustrated is the system used to construct the IgH recombinants. In the case of constructing the Ter3/Ter462* double mutant, the pTer462* vector was cut at the indicated NdeI sites, and the Cμ-gpt–containing fragment was purified by electrophoresis. In the case of constructing the Ter3 recombinant, the pTer462* vector was simply linearized at the MluI site. The DNA was introduced into the Ter3 cell line by electroporation and plated in MHX medium at limiting dilution, as described (Oancea and Shulman, 1994; Buzina and Shulman, 1996). MHX^R (gpt⁺) transformants were tested for the replacement of the Cμ region by amplifying the indicated segment by PCR and testing its sensitivity to XmnI. We then tested for proper targeting by amplifying the 3′ junction segment by PCR, as illustrated.