FIGURE 1:

Generation of the Lmnb1^B2 allele, which yields lamin B2 from the Lmnb1 locus. (A) Map of the Lmnb1 locus and the targeting vector, which was designed to introduce a Lmnb2 cDNA into the translational start site in exon 1 of Lmnb1 (at an NcoI site). The Lmnb2 cDNA was modified to introduce a new EcoRV site and remove an existing SacI site (depicted by an asterisk), making it possible to distinguish Lmnb1^B2 transcripts from those of the endogenous Lmnb2 locus. Exons are depicted as black boxes (E1 and E2); the noncoding region of exon 1 is in white. Black arrowheads indicate the loxP sites. The neo cassette is shown as a gray box; a diphtheria toxin (DTA) counterselection cassette is shown as a black box. The primers used for recombineering (GF, GR, LF, and LR) are indicated. The primers used for 5′ long-range PCR (5′ LR-PCR) and 3′ long-range PCR (3′ LR-PCR) are indicated by arrows. (B) Screening of ES cell clones by 5′ long-range PCR. A 5.8-kb fragment was amplified from the Lmnb1^B2 allele; the identity of the fragment was confirmed by EcoRV digestion (yielding 5.4-and 0.4-kb fragments). (C) Screening of ES cell clones by 3′ long-range PCR. A 6-kb DNA fragment was amplified from the Lmnb1^B2 allele; the identity of the fragment was confirmed with BamHI digestion (yielding 4.3- and 1.7-kb fragments). A nonspecific band is indicated by an asterisk. (D) EcoRV digestion of a 950–base pair Lmnb2 RT-PCR fragment (amplicon from exons 1–7 of Lmnb2) from Lmnb1^B2/+ and Lmnb1^+/+ ES cells. Lmnb2 RT-PCR DNA fragments from the Lmnb1^B2 allele (but not from the endogenous Lmnb2 allele) were cleaved by EcoRV (yielding an 850–base pair fragment). (E) DNA sequencing chromatogram of an RT-PCR fragment from the Lmnb1^B2 allele showing the junction between the Lmnb1 5′ UTR and Lmnb2 coding DNA sequences (CDS).