Figure 1
Figure 1. Germ line gene disruption generates a loss-of-expression PARP-14 allele in mice. (A) Western blot analysis of CoaSt6/PARP-14 levels in T and B lymphocytes. Extracts from T and B lymphocytes, purified as described in “Northern blot RT-PCR,” were subjected to immunoblot analysis using affinity-purified antipeptide antibodies against CoaSt6 (PARP-14) or actin, as indicated. (B) Equal masses of protein in extracts from normal B cells or carefully excised primary tumor masses from 4 separate mice (1-4) were analyzed by Western blotting as described in panel A. Shown to the right is a bar graph of the results from quantitating the fluorescence intensity of the Western bands and normalizing to actin (PARP-14 was reproducibly increased with or without normalization). Similar results were obtained with cultured B-lymphoma lines (Figure S1). (C) Simplified map schematizing the normal and mutated PARP-14 alleles, EcoR1 restriction sites used for Southern blot analysis, and positions of hybridization probe and PCR primers used. Black boxes represent exons 1 to 4 and 8, E1 to E4 and E8, respectively; other portions of the locus downstream from intron 4 are omitted. Gene expression was disrupted by insertion of a splice-trap element containing an EcoR1 site; PARP-14 PCR primers flanked the insertion site in intron 1 (I1), which along with a splice-trap specific primer permitted unambiguous assignment of the allele on each chromosome. E8 primers used for RT-PCR also are diagrammed (sequences in Table S1). (D) Descendants were bred from founder lines established as described in “Generation of PARP-14–deficient mice.” Shown is an autoradiograph of Southern blots of EcoR1-digested genomic DNA from mice of indicated genotype (identified by PCR using the indicated I1-specific primers, sequences in Table S1) after probing with radiolabeled PARP-14 5′ sequences (gray rectangle in panel C) to reveal approximately 7.3-kb and approximately 5.7-kb bands (WT and mutated, respectively). (E) Total RNA isolated from thymus or lung of PARP-14+/+, PARP-14+/−, and PARP-14−/− mice, analyzed by Northern blots probed with radiolabeled PARP-14 (551 bp) or GAPDH DNA fragments. (F) RT-PCR using RNA isolated from lymphoid tissues of +/+, +/−, and −/− mice. The primer pair (downstream pair in panel C) detects sequences downstream from the gene disruption. (G) Immunoblot analysis of BAL2b/CoaSt6/PARP-14 and actin in splenocytes from WT and PARP-14−/− mice. Similar data were obtained with extracts of lung, and analysis of the full gel lane disclosed no evidence of a truncated form of the polypeptide.

Germ line gene disruption generates a loss-of-expression PARP-14 allele in mice. (A) Western blot analysis of CoaSt6/PARP-14 levels in T and B lymphocytes. Extracts from T and B lymphocytes, purified as described in “Northern blot RT-PCR,” were subjected to immunoblot analysis using affinity-purified antipeptide antibodies against CoaSt6 (PARP-14) or actin, as indicated. (B) Equal masses of protein in extracts from normal B cells or carefully excised primary tumor masses from 4 separate mice (1-4) were analyzed by Western blotting as described in panel A. Shown to the right is a bar graph of the results from quantitating the fluorescence intensity of the Western bands and normalizing to actin (PARP-14 was reproducibly increased with or without normalization). Similar results were obtained with cultured B-lymphoma lines (Figure S1). (C) Simplified map schematizing the normal and mutated PARP-14 alleles, EcoR1 restriction sites used for Southern blot analysis, and positions of hybridization probe and PCR primers used. Black boxes represent exons 1 to 4 and 8, E1 to E4 and E8, respectively; other portions of the locus downstream from intron 4 are omitted. Gene expression was disrupted by insertion of a splice-trap element containing an EcoR1 site; PARP-14 PCR primers flanked the insertion site in intron 1 (I1), which along with a splice-trap specific primer permitted unambiguous assignment of the allele on each chromosome. E8 primers used for RT-PCR also are diagrammed (sequences in Table S1). (D) Descendants were bred from founder lines established as described in “Generation of PARP-14–deficient mice.” Shown is an autoradiograph of Southern blots of EcoR1-digested genomic DNA from mice of indicated genotype (identified by PCR using the indicated I1-specific primers, sequences in Table S1) after probing with radiolabeled PARP-14 5′ sequences (gray rectangle in panel C) to reveal approximately 7.3-kb and approximately 5.7-kb bands (WT and mutated, respectively). (E) Total RNA isolated from thymus or lung of PARP-14+/+, PARP-14+/−, and PARP-14−/− mice, analyzed by Northern blots probed with radiolabeled PARP-14 (551 bp) or GAPDH DNA fragments. (F) RT-PCR using RNA isolated from lymphoid tissues of +/+, +/−, and −/− mice. The primer pair (downstream pair in panel C) detects sequences downstream from the gene disruption. (G) Immunoblot analysis of BAL2b/CoaSt6/PARP-14 and actin in splenocytes from WT and PARP-14−/− mice. Similar data were obtained with extracts of lung, and analysis of the full gel lane disclosed no evidence of a truncated form of the polypeptide.

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