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Impairment of T cell development in deltaEF1 mutant mice.

Higashi Y, Moribe H, Takagi T, Sekido R, Kawakami K, Kikutani H, Kondoh H - J. Exp. Med. (1997)

Bottom Line: Analysis of the mutant thymocyte showed reduction of the total cell number by two orders of magnitude accompanying the impaired thymocyte development.In contrast to T cells, other hematopoietic lineages appeared to be normal.The data indicated that deltaEF1 is involved in regulation of T cell development at multiple stages.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular and Cellular Biology, Osaka University, Suita, Japan.

ABSTRACT
Using the method of gene targeting in mouse embryonic stem cells, regulatory function of deltaEF1, a zinc finger and homeodomain-containing transcription factor, was investigated in vivo by generating the deltaEF1 mutant mice. The mutated allele of deltaEF1 produced a truncated form of the deltaEF1 protein lacking a zinc finger cluster proximal to COOH terminus. The homozygous deltaEF1 mutant mice had poorly developed thymi with no distinction of cortex and medulla. Analysis of the mutant thymocyte showed reduction of the total cell number by two orders of magnitude accompanying the impaired thymocyte development. The early stage intrathymic c-kit+ T precursor cells were largely depleted. The following thymocyte development also seemed to be affected as assessed by the distorted composition of CD4- or CD8-expressing cells. The mutant thymocyte showed elevated alpha4 integrin expression, which might be related to the T cell defect in the mutant mice. In the peripheral lymph node tissue of the mutant mice, the CD4-CD8+ single positive cells were significantly reduced relative to CD4+CD8-single positive cells. In contrast to T cells, other hematopoietic lineages appeared to be normal. The data indicated that deltaEF1 is involved in regulation of T cell development at multiple stages.

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Related in: MedlinePlus

ΔC-fin δEF1 mutant allele generated by homologous recombination.  (A) The last  three exons (6–8) of the mouse δEF1 gene encoding the homeodomain and the C-proximal zinc  finger cluster are shown (top) together with the targeting vector (middle) and the resulting genomic structure of the homologous recombinant (bottom). Stop codons and the neor cassette are  inserted in the middle of the 6th exon, downstream of the homeodomain in the targeting vector. A DT-A cassette (11) was added at the 3′ end of the vector for negative selection against  random insertion of the vector. neor, neomycin resistance gene cassette; DT-A, the Diphtheria  toxin A chain expression cassette. The diagnostic BglII fragments detected in Southern blots using the probe (indicated by the thick bar) are shown. Restriction sites of SalI, ApaI, and Sau3AI  in the genomic DNA used for the targeting vector construction are also shown (see Materials  and Methods). (B) Proteins coded by wild-type allele (wt) and the mutated (ΔC-fin) allele are  schematically shown. (C) DNAs isolated from wild-type mice (+/+), a recombinant ES clone  (A84), and mice heterozygous (+/−) or homozygous (−/−) for the mutant δEF1 gene were  digested with BglII and subjected to Southern blot analysis using the indicated probe. (D) Total  RNAs (5 μg each) from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) embryos (12.5 d.p.c.) were analyzed by Northern blotting using a mouse δEF1 cDNA (clone  M12) as probe. Only the larger size mRNA (δEF1+neor) resulting from the insertion of neor  was detected in a homozygous embryo, while only the normal size of δEF1 mRNA was present  in a wild type, and both were in a heterozygous embryo. (E) Nuclear extracts from wild-type  (+/+), heterozygous (+/−) and homozygous (−/−) 12.5 d.p.c. embryos were immunoprecipitated and analyzed by Western blotting for δEF1 and \xc6 C-fin protein using anti-δEF1 antiserum which can react to N-proximal portion of δEF1 (see Materials and Methods). As size references, the nuclear extracts from the COS cells transfected with expression vectors of wild-type  (wt/COS) and ΔC-fin δEF1 protein (ΔC-fin/COS) were also electrophoresed in parallel.
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Figure 2: ΔC-fin δEF1 mutant allele generated by homologous recombination. (A) The last three exons (6–8) of the mouse δEF1 gene encoding the homeodomain and the C-proximal zinc finger cluster are shown (top) together with the targeting vector (middle) and the resulting genomic structure of the homologous recombinant (bottom). Stop codons and the neor cassette are inserted in the middle of the 6th exon, downstream of the homeodomain in the targeting vector. A DT-A cassette (11) was added at the 3′ end of the vector for negative selection against random insertion of the vector. neor, neomycin resistance gene cassette; DT-A, the Diphtheria toxin A chain expression cassette. The diagnostic BglII fragments detected in Southern blots using the probe (indicated by the thick bar) are shown. Restriction sites of SalI, ApaI, and Sau3AI in the genomic DNA used for the targeting vector construction are also shown (see Materials and Methods). (B) Proteins coded by wild-type allele (wt) and the mutated (ΔC-fin) allele are schematically shown. (C) DNAs isolated from wild-type mice (+/+), a recombinant ES clone (A84), and mice heterozygous (+/−) or homozygous (−/−) for the mutant δEF1 gene were digested with BglII and subjected to Southern blot analysis using the indicated probe. (D) Total RNAs (5 μg each) from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) embryos (12.5 d.p.c.) were analyzed by Northern blotting using a mouse δEF1 cDNA (clone M12) as probe. Only the larger size mRNA (δEF1+neor) resulting from the insertion of neor was detected in a homozygous embryo, while only the normal size of δEF1 mRNA was present in a wild type, and both were in a heterozygous embryo. (E) Nuclear extracts from wild-type (+/+), heterozygous (+/−) and homozygous (−/−) 12.5 d.p.c. embryos were immunoprecipitated and analyzed by Western blotting for δEF1 and \xc6 C-fin protein using anti-δEF1 antiserum which can react to N-proximal portion of δEF1 (see Materials and Methods). As size references, the nuclear extracts from the COS cells transfected with expression vectors of wild-type (wt/COS) and ΔC-fin δEF1 protein (ΔC-fin/COS) were also electrophoresed in parallel.

Mentions: Cloning and structural analysis of mouse δEF1 has been described (9). The targeting vector (see Fig. 2 A) was constructed as follows. A 0.8-kb SalI–SalI fragment containing a Sau3AI–SalI genomic fragment of the exon 6 sequence (see Fig. 2 A) and a 12-bp SalI–BamHI adapter sequence which is derived from an EMBL3 cloning vector, was subcloned into the SalI site of pBlueScript II to give pSS. The SalI and XbaI sites at the 5′ end of the inserted genomic fragment of pSS were inactivated by digesting with XbaI, partially with SalI, blunt-ending by fill-in, and self-ligation. A XbaI linker carrying stop codons in all three frames (CTAGTCTAGACTAG) was inserted in the remaining SalI site at the 3′ end of the insert to have pSSstop. A XhoI–KpnI fragment of pSTNeoB (10) containing neor sequence was inserted in the XhoI–KpnI site of the pSSstop, to have pSSstopNEO. In parallel, the 5.4-kb SalI-ApaI genomic fragment, immediately 3′ of the Sau3AI–SalI fragment was once cloned into the pBlueScript II, and regenerated by digesting with Asp718. The resulting fragment was blunt-ended, digested with SalI and cloned into the SalI–EcoRV site of DT-A vector (11), generating pSADT-A plasmid. The pSSstopNEO was digested with the Asp718, blunt-ended and digested with NotI. The resulting Asp718 (blunt-ended by Klenow)-NotI fragment was cloned into the SalI (blunt-ended by Klenow)–NotI sites of the pSADT-A, generating a final targeting vector. The vector plasmid was linearized with NotI and used for electroporation. The expected targeted gene product lacks the COOH-terminal zinc finger clusters which have been shown to be essential for the DNA binding of δEF1 protein (12). The neor element has a promoter but lacks the termination and poly(A) addition signals, so that the neor is expressed only when poly(A) addition signal is supplied by recombination with a host gene. A DT-A cassette (11) was placed in the 3′ end of the linearized vector for the negative selection against integration into nonhomologous genes.


Impairment of T cell development in deltaEF1 mutant mice.

Higashi Y, Moribe H, Takagi T, Sekido R, Kawakami K, Kikutani H, Kondoh H - J. Exp. Med. (1997)

ΔC-fin δEF1 mutant allele generated by homologous recombination.  (A) The last  three exons (6–8) of the mouse δEF1 gene encoding the homeodomain and the C-proximal zinc  finger cluster are shown (top) together with the targeting vector (middle) and the resulting genomic structure of the homologous recombinant (bottom). Stop codons and the neor cassette are  inserted in the middle of the 6th exon, downstream of the homeodomain in the targeting vector. A DT-A cassette (11) was added at the 3′ end of the vector for negative selection against  random insertion of the vector. neor, neomycin resistance gene cassette; DT-A, the Diphtheria  toxin A chain expression cassette. The diagnostic BglII fragments detected in Southern blots using the probe (indicated by the thick bar) are shown. Restriction sites of SalI, ApaI, and Sau3AI  in the genomic DNA used for the targeting vector construction are also shown (see Materials  and Methods). (B) Proteins coded by wild-type allele (wt) and the mutated (ΔC-fin) allele are  schematically shown. (C) DNAs isolated from wild-type mice (+/+), a recombinant ES clone  (A84), and mice heterozygous (+/−) or homozygous (−/−) for the mutant δEF1 gene were  digested with BglII and subjected to Southern blot analysis using the indicated probe. (D) Total  RNAs (5 μg each) from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) embryos (12.5 d.p.c.) were analyzed by Northern blotting using a mouse δEF1 cDNA (clone  M12) as probe. Only the larger size mRNA (δEF1+neor) resulting from the insertion of neor  was detected in a homozygous embryo, while only the normal size of δEF1 mRNA was present  in a wild type, and both were in a heterozygous embryo. (E) Nuclear extracts from wild-type  (+/+), heterozygous (+/−) and homozygous (−/−) 12.5 d.p.c. embryos were immunoprecipitated and analyzed by Western blotting for δEF1 and \xc6 C-fin protein using anti-δEF1 antiserum which can react to N-proximal portion of δEF1 (see Materials and Methods). As size references, the nuclear extracts from the COS cells transfected with expression vectors of wild-type  (wt/COS) and ΔC-fin δEF1 protein (ΔC-fin/COS) were also electrophoresed in parallel.
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Related In: Results  -  Collection

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Figure 2: ΔC-fin δEF1 mutant allele generated by homologous recombination. (A) The last three exons (6–8) of the mouse δEF1 gene encoding the homeodomain and the C-proximal zinc finger cluster are shown (top) together with the targeting vector (middle) and the resulting genomic structure of the homologous recombinant (bottom). Stop codons and the neor cassette are inserted in the middle of the 6th exon, downstream of the homeodomain in the targeting vector. A DT-A cassette (11) was added at the 3′ end of the vector for negative selection against random insertion of the vector. neor, neomycin resistance gene cassette; DT-A, the Diphtheria toxin A chain expression cassette. The diagnostic BglII fragments detected in Southern blots using the probe (indicated by the thick bar) are shown. Restriction sites of SalI, ApaI, and Sau3AI in the genomic DNA used for the targeting vector construction are also shown (see Materials and Methods). (B) Proteins coded by wild-type allele (wt) and the mutated (ΔC-fin) allele are schematically shown. (C) DNAs isolated from wild-type mice (+/+), a recombinant ES clone (A84), and mice heterozygous (+/−) or homozygous (−/−) for the mutant δEF1 gene were digested with BglII and subjected to Southern blot analysis using the indicated probe. (D) Total RNAs (5 μg each) from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) embryos (12.5 d.p.c.) were analyzed by Northern blotting using a mouse δEF1 cDNA (clone M12) as probe. Only the larger size mRNA (δEF1+neor) resulting from the insertion of neor was detected in a homozygous embryo, while only the normal size of δEF1 mRNA was present in a wild type, and both were in a heterozygous embryo. (E) Nuclear extracts from wild-type (+/+), heterozygous (+/−) and homozygous (−/−) 12.5 d.p.c. embryos were immunoprecipitated and analyzed by Western blotting for δEF1 and \xc6 C-fin protein using anti-δEF1 antiserum which can react to N-proximal portion of δEF1 (see Materials and Methods). As size references, the nuclear extracts from the COS cells transfected with expression vectors of wild-type (wt/COS) and ΔC-fin δEF1 protein (ΔC-fin/COS) were also electrophoresed in parallel.
Mentions: Cloning and structural analysis of mouse δEF1 has been described (9). The targeting vector (see Fig. 2 A) was constructed as follows. A 0.8-kb SalI–SalI fragment containing a Sau3AI–SalI genomic fragment of the exon 6 sequence (see Fig. 2 A) and a 12-bp SalI–BamHI adapter sequence which is derived from an EMBL3 cloning vector, was subcloned into the SalI site of pBlueScript II to give pSS. The SalI and XbaI sites at the 5′ end of the inserted genomic fragment of pSS were inactivated by digesting with XbaI, partially with SalI, blunt-ending by fill-in, and self-ligation. A XbaI linker carrying stop codons in all three frames (CTAGTCTAGACTAG) was inserted in the remaining SalI site at the 3′ end of the insert to have pSSstop. A XhoI–KpnI fragment of pSTNeoB (10) containing neor sequence was inserted in the XhoI–KpnI site of the pSSstop, to have pSSstopNEO. In parallel, the 5.4-kb SalI-ApaI genomic fragment, immediately 3′ of the Sau3AI–SalI fragment was once cloned into the pBlueScript II, and regenerated by digesting with Asp718. The resulting fragment was blunt-ended, digested with SalI and cloned into the SalI–EcoRV site of DT-A vector (11), generating pSADT-A plasmid. The pSSstopNEO was digested with the Asp718, blunt-ended and digested with NotI. The resulting Asp718 (blunt-ended by Klenow)-NotI fragment was cloned into the SalI (blunt-ended by Klenow)–NotI sites of the pSADT-A, generating a final targeting vector. The vector plasmid was linearized with NotI and used for electroporation. The expected targeted gene product lacks the COOH-terminal zinc finger clusters which have been shown to be essential for the DNA binding of δEF1 protein (12). The neor element has a promoter but lacks the termination and poly(A) addition signals, so that the neor is expressed only when poly(A) addition signal is supplied by recombination with a host gene. A DT-A cassette (11) was placed in the 3′ end of the linearized vector for the negative selection against integration into nonhomologous genes.

Bottom Line: Analysis of the mutant thymocyte showed reduction of the total cell number by two orders of magnitude accompanying the impaired thymocyte development.In contrast to T cells, other hematopoietic lineages appeared to be normal.The data indicated that deltaEF1 is involved in regulation of T cell development at multiple stages.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular and Cellular Biology, Osaka University, Suita, Japan.

ABSTRACT
Using the method of gene targeting in mouse embryonic stem cells, regulatory function of deltaEF1, a zinc finger and homeodomain-containing transcription factor, was investigated in vivo by generating the deltaEF1 mutant mice. The mutated allele of deltaEF1 produced a truncated form of the deltaEF1 protein lacking a zinc finger cluster proximal to COOH terminus. The homozygous deltaEF1 mutant mice had poorly developed thymi with no distinction of cortex and medulla. Analysis of the mutant thymocyte showed reduction of the total cell number by two orders of magnitude accompanying the impaired thymocyte development. The early stage intrathymic c-kit+ T precursor cells were largely depleted. The following thymocyte development also seemed to be affected as assessed by the distorted composition of CD4- or CD8-expressing cells. The mutant thymocyte showed elevated alpha4 integrin expression, which might be related to the T cell defect in the mutant mice. In the peripheral lymph node tissue of the mutant mice, the CD4-CD8+ single positive cells were significantly reduced relative to CD4+CD8-single positive cells. In contrast to T cells, other hematopoietic lineages appeared to be normal. The data indicated that deltaEF1 is involved in regulation of T cell development at multiple stages.

Show MeSH
Related in: MedlinePlus