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Joining chain-expressing and -nonexpressing B cell populations in the mouse.

Erlandsson L, Akerblad P, Vingsbo-Lundberg C, Kallberg E, Lycke N, Leanderson T - J. Exp. Med. (2001)

Bottom Line: Serum immunoglobulin (Ig)M and serum IgG were reduced six to eightfold, while serum IgA was elevated 14-fold in these mice.Analysis of single B cells stimulated by lipopolysaccharide in vitro using reverse transcription-polymerase chain reaction showed that J chain-nonexpressing B cells could be detected that had a secretory phenotype as determined by an abundance of transcript for secretory IgM.Finally, limiting dilution analysis of peripheral B cells showed that J chain expression was a clonal property already established in naive, peripheral B lymphocytes.

View Article: PubMed Central - PubMed

Affiliation: Immunology Section, Department of Cell and Molecular Biology, Lund University, S-221 84 Lund, Sweden.

ABSTRACT
The diphtheria toxin A chain (DTA) was gene targeted into the Joining chain (J chain) locus to create a mouse strain selecting against J chain-expressing cells, JDTA mice. Serum immunoglobulin (Ig)M and serum IgG were reduced six to eightfold, while serum IgA was elevated 14-fold in these mice. JDTA mice were immune competent although the serum Ig response compared with wild-type mice was reduced sixfold at day 14 but only fourfold at day 45 after immunization. Exchanging the DTA gene with a cDNA for c-myc resulted in mice with a distinct phenotype with increased Ig production and enhanced humoral immune responses. Analysis of single B cells stimulated by lipopolysaccharide in vitro using reverse transcription-polymerase chain reaction showed that J chain-nonexpressing B cells could be detected that had a secretory phenotype as determined by an abundance of transcript for secretory IgM. Finally, limiting dilution analysis of peripheral B cells showed that J chain expression was a clonal property already established in naive, peripheral B lymphocytes.

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Replacement of the J chain exon 1 with the DTA gene. (A) Disruption of the J chain gene and gene replacement by homologous recombination. Genomic map of J chain locus before and after homologous recombination. Digestion with KpnI gives an endogenous band of 5.6 kb while the recombined locus gives a band of 8.0 kb for JDTAneo and 6.4 kb for JDTA after removal of the neor gene. Southern blot analysis of the recombined J chain locus showing wt, heterozygous (+/−) JDTAneo, and heterozygous (+/−) JDTA using an outside probe (not present in the construct used for targeting). E, EcoRI; B, BamHI; S, SacI; P, PstI; K, KpnI; and H, HindIII. (B) Expression analysis by RT-PCR for J chain and DTA transcripts in wt, JDTA heterozygous (JDTA+/−), and JDTA mice. Primers for J chain were located in exon 1 and in exon 4 of the J chain locus. Primers for DTA transcripts were located in the 3′ end of the DTA gene and in the exon 4 of the J chain locus. Shown is also analysis for HPRT transcripts as a cDNA control.
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Figure 1: Replacement of the J chain exon 1 with the DTA gene. (A) Disruption of the J chain gene and gene replacement by homologous recombination. Genomic map of J chain locus before and after homologous recombination. Digestion with KpnI gives an endogenous band of 5.6 kb while the recombined locus gives a band of 8.0 kb for JDTAneo and 6.4 kb for JDTA after removal of the neor gene. Southern blot analysis of the recombined J chain locus showing wt, heterozygous (+/−) JDTAneo, and heterozygous (+/−) JDTA using an outside probe (not present in the construct used for targeting). E, EcoRI; B, BamHI; S, SacI; P, PstI; K, KpnI; and H, HindIII. (B) Expression analysis by RT-PCR for J chain and DTA transcripts in wt, JDTA heterozygous (JDTA+/−), and JDTA mice. Primers for J chain were located in exon 1 and in exon 4 of the J chain locus. Primers for DTA transcripts were located in the 3′ end of the DTA gene and in the exon 4 of the J chain locus. Shown is also analysis for HPRT transcripts as a cDNA control.

Mentions: The mouse J chain gene locus was cloned from an I129 genomic phage library and a targeting vector for gene replacement was constructed as shown in Fig. 1 A and 6 A. The diphtheria toxin A chain (DTA) subunit in a pUC plasmid 41 was given to us by Ian H. Maxwell, University of Colorado Health Services Center, Denver, Colorado. A 795-bp BglII fragment containing the DTA gene (without its polyA sequence) or a 1.4-kb murine c-myc cDNA fragment (a gift from Dr. Richard H. Scheuermann, University of Texas) was cloned 3′ of a 5-kb genomic fragment containing the J chain promoter. An EcoRI/XhoI fragment from the ploxPneo-1 plasmid containing the neomycin resistance gene (neor) under the control of the phosphoglycerate kinase promoter and flanked by loxP sites was cloned 3′ of the DTA gene. A J chain splice site was synthesized and cloned 3′ of the neor gene after which a 10-kb KpnI/SalI fragment 3′ of exon 1 was cloned. The result was a construct where exon 1 of the J chain gene was replaced by the DTA or the cMyc together with the neor gene. The phosphoglycerate kinase promoter Herpes simplex thymidine kinase gene was cloned into the polylinker 3′ of the J chain construct. The whole construct was linearized by a unique NotI site 3′ of the thymidine kinase gene and transfected into the embryonic stem (ES) cell line E14 provided by Dr. Werner Muller, Institute for Genetics, Cologne, Germany. Colonies were screened by Southern blotting for homologous recombination events after KpnI digestion and probed with an outside probe (see Fig. 1 A and 5 A). A targeted JDTAneo clone was subsequently transiently transfected with the plasmid pBS.Cre provided by Dr. Reinhard Fassler, Lund University, to remove the neor gene, leaving only one loxP sequence in the locus. Colonies that had lost their resistance to G418 were expanded, DNA prepared and screened by Southern blotting for removal of the neor gene (see Fig. 1 A). A targeted JcMycneo clone was used to generate a mouse strain which subsequently was bred to mice constitutively expressing the Cre enzyme 42, enabling in vivo excision of the neor gene. The thereby generated JcMyc mice were identified by Southern blot analysis on DNA from tail biopsies (see Fig. 5 A).


Joining chain-expressing and -nonexpressing B cell populations in the mouse.

Erlandsson L, Akerblad P, Vingsbo-Lundberg C, Kallberg E, Lycke N, Leanderson T - J. Exp. Med. (2001)

Replacement of the J chain exon 1 with the DTA gene. (A) Disruption of the J chain gene and gene replacement by homologous recombination. Genomic map of J chain locus before and after homologous recombination. Digestion with KpnI gives an endogenous band of 5.6 kb while the recombined locus gives a band of 8.0 kb for JDTAneo and 6.4 kb for JDTA after removal of the neor gene. Southern blot analysis of the recombined J chain locus showing wt, heterozygous (+/−) JDTAneo, and heterozygous (+/−) JDTA using an outside probe (not present in the construct used for targeting). E, EcoRI; B, BamHI; S, SacI; P, PstI; K, KpnI; and H, HindIII. (B) Expression analysis by RT-PCR for J chain and DTA transcripts in wt, JDTA heterozygous (JDTA+/−), and JDTA mice. Primers for J chain were located in exon 1 and in exon 4 of the J chain locus. Primers for DTA transcripts were located in the 3′ end of the DTA gene and in the exon 4 of the J chain locus. Shown is also analysis for HPRT transcripts as a cDNA control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195946&req=5

Figure 1: Replacement of the J chain exon 1 with the DTA gene. (A) Disruption of the J chain gene and gene replacement by homologous recombination. Genomic map of J chain locus before and after homologous recombination. Digestion with KpnI gives an endogenous band of 5.6 kb while the recombined locus gives a band of 8.0 kb for JDTAneo and 6.4 kb for JDTA after removal of the neor gene. Southern blot analysis of the recombined J chain locus showing wt, heterozygous (+/−) JDTAneo, and heterozygous (+/−) JDTA using an outside probe (not present in the construct used for targeting). E, EcoRI; B, BamHI; S, SacI; P, PstI; K, KpnI; and H, HindIII. (B) Expression analysis by RT-PCR for J chain and DTA transcripts in wt, JDTA heterozygous (JDTA+/−), and JDTA mice. Primers for J chain were located in exon 1 and in exon 4 of the J chain locus. Primers for DTA transcripts were located in the 3′ end of the DTA gene and in the exon 4 of the J chain locus. Shown is also analysis for HPRT transcripts as a cDNA control.
Mentions: The mouse J chain gene locus was cloned from an I129 genomic phage library and a targeting vector for gene replacement was constructed as shown in Fig. 1 A and 6 A. The diphtheria toxin A chain (DTA) subunit in a pUC plasmid 41 was given to us by Ian H. Maxwell, University of Colorado Health Services Center, Denver, Colorado. A 795-bp BglII fragment containing the DTA gene (without its polyA sequence) or a 1.4-kb murine c-myc cDNA fragment (a gift from Dr. Richard H. Scheuermann, University of Texas) was cloned 3′ of a 5-kb genomic fragment containing the J chain promoter. An EcoRI/XhoI fragment from the ploxPneo-1 plasmid containing the neomycin resistance gene (neor) under the control of the phosphoglycerate kinase promoter and flanked by loxP sites was cloned 3′ of the DTA gene. A J chain splice site was synthesized and cloned 3′ of the neor gene after which a 10-kb KpnI/SalI fragment 3′ of exon 1 was cloned. The result was a construct where exon 1 of the J chain gene was replaced by the DTA or the cMyc together with the neor gene. The phosphoglycerate kinase promoter Herpes simplex thymidine kinase gene was cloned into the polylinker 3′ of the J chain construct. The whole construct was linearized by a unique NotI site 3′ of the thymidine kinase gene and transfected into the embryonic stem (ES) cell line E14 provided by Dr. Werner Muller, Institute for Genetics, Cologne, Germany. Colonies were screened by Southern blotting for homologous recombination events after KpnI digestion and probed with an outside probe (see Fig. 1 A and 5 A). A targeted JDTAneo clone was subsequently transiently transfected with the plasmid pBS.Cre provided by Dr. Reinhard Fassler, Lund University, to remove the neor gene, leaving only one loxP sequence in the locus. Colonies that had lost their resistance to G418 were expanded, DNA prepared and screened by Southern blotting for removal of the neor gene (see Fig. 1 A). A targeted JcMycneo clone was used to generate a mouse strain which subsequently was bred to mice constitutively expressing the Cre enzyme 42, enabling in vivo excision of the neor gene. The thereby generated JcMyc mice were identified by Southern blot analysis on DNA from tail biopsies (see Fig. 5 A).

Bottom Line: Serum immunoglobulin (Ig)M and serum IgG were reduced six to eightfold, while serum IgA was elevated 14-fold in these mice.Analysis of single B cells stimulated by lipopolysaccharide in vitro using reverse transcription-polymerase chain reaction showed that J chain-nonexpressing B cells could be detected that had a secretory phenotype as determined by an abundance of transcript for secretory IgM.Finally, limiting dilution analysis of peripheral B cells showed that J chain expression was a clonal property already established in naive, peripheral B lymphocytes.

View Article: PubMed Central - PubMed

Affiliation: Immunology Section, Department of Cell and Molecular Biology, Lund University, S-221 84 Lund, Sweden.

ABSTRACT
The diphtheria toxin A chain (DTA) was gene targeted into the Joining chain (J chain) locus to create a mouse strain selecting against J chain-expressing cells, JDTA mice. Serum immunoglobulin (Ig)M and serum IgG were reduced six to eightfold, while serum IgA was elevated 14-fold in these mice. JDTA mice were immune competent although the serum Ig response compared with wild-type mice was reduced sixfold at day 14 but only fourfold at day 45 after immunization. Exchanging the DTA gene with a cDNA for c-myc resulted in mice with a distinct phenotype with increased Ig production and enhanced humoral immune responses. Analysis of single B cells stimulated by lipopolysaccharide in vitro using reverse transcription-polymerase chain reaction showed that J chain-nonexpressing B cells could be detected that had a secretory phenotype as determined by an abundance of transcript for secretory IgM. Finally, limiting dilution analysis of peripheral B cells showed that J chain expression was a clonal property already established in naive, peripheral B lymphocytes.

Show MeSH
Related in: MedlinePlus