Two genetically separable steps in the differentiation of thymic epithelium

Science

Volumn 272, Issue 5263, 1996, Pages 886-889

  Nehls, Michael ^a,c   Kyewski, Bruno ^a   Messerle, Martin ^a,d   Waldschütz, Ralph ^a   Schüddekopf, Kerstin ^a,e   Smith, Andrew J H ^b,f   Boehm, Thomas ^a  

^a GERMAN CANCER RESEARCH CENTER DKFZ   (Germany)

^b MRC LABORATORY OF MOLECULAR BIOLOGY   (United Kingdom)

^c GOETHE UNIVERSITY   (Germany)

^d UNIVERSITY OF HEIDELBERG   (Germany)

^e UNIVERSITY OF HAMBURG   (Germany)

^f UNIVERSITY OF EDINBURGH   (United Kingdom)

Author keywords

[No Author keywords available]

Indexed keywords

TRANSCRIPTION FACTOR;

ANIMAL CELL; ANIMAL EXPERIMENT; ARTICLE; CELL DIFFERENTIATION; CONTROLLED STUDY; EMBRYO; EPITHELIUM CELL; GENE ACTIVITY; GENE LOCUS; GENE MUTATION; MESENCHYME; MOUSE; NEWBORN; NONHUMAN; PHENOTYPE; PRIORITY JOURNAL; PROLYMPHOCYTE; THYMOCYTE;

ANIMALIA; MUS MUSCULUS;

EID: 0029920145     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.272.5263.886     Document Type: Article

References (34)

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18. β-Galactosidase activity correlates with the transcriptional activity of the targeted gene (9); because no endogenous β-galactosidase activity can be detected in wild-type thymuses at all stages of development, whn gene expression during thymic development was characterized throughout with the β-galactosidase reporter protein.
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24. 2, 0.04% X-Gal, 5 mM potassium ferrocyanide, and 5 mM ferricyanide. Tissues were then washed, post-fixed in PBS and 4% paraformaldehyde, and processed for histological analysis. Sections were counterstained with eosin and hematoxylin.
25. The complete genomic structure of the mouse whn locus will be described (M. Schorpp et al., in preparation).
26. The targeting construct was derived as follows. P1 recombinants spanning the whn locus [M. Nehls et al., Mamm. Genome 6, 321 (1995)] were partially digested with Sau 3Al, selected by size, and cloned into XKO [M. Nehls, M. Messerle, A. Sirulnik, A. J. H. Smith, T. Boehm, Biotechniques 17, 770 (1994)], which supplies thymidine kinase genes at either end of the insert. Recombinants containing exon 3 were analyzed for inserts in which the Sfi I site was at a reasonable distance from one end of the insert, to facilitate subsequent polymerase chain reaction (PCR) screening. In the clone used here (32), the Sfi I site was located about 1.8 kb downstream of the 5′ end of the homologous sequences and about 4.2 kb upstream of the 3′ end of the homologous sequences. Phage DNA was concatamerized by ligation at its cos ends and then cut with Sfi I. The Sfi I ends were modified by addition of Bam HI adapter oligonucleotides and this DNA subsequently ligated to a Bam HI cassette containing a promoterless β-galactosidase gene preceded by an internal ribosomal entry sequence followed by a neomycin resistance gene (7). The ligation was packaged in vitro, and desired recombinants were selected by plaque hybridization, converted into plasmid form, and linearized with Not I. This linear molecule was then ligated to a self-complementary Not I-compatible, phosphorylated oligonucleotide (5′-GGCCTCCGGTACATGATCGAGGGGACTGACAAGACGGCCAGTCCTCGATCATGTACCGGA-3′) to seal the ends of the linear fragment. This treatment enhances the stability of transfected DNA, improves positive-negative selection, and increases the frequency of homologous recombination (M. Messerle and T. Boehm, in preparation). The targeting construct was transfected into CJ7 cells [P. J. Swiatek and T. Gridley, Genes Dev. 7, 2071 (1993)], and clones resistant to G418 and gancyclovir were analyzed by PCR and Southern (DNA) blotting for homologous recombination. Of 25 colonies tested, 23 were homologous integrants. Mice heterozygous for the insertional mutation were derived as described [A. Warren et al., Cell 15, 45 (1994)].
27. The targeting construct was derived as follows. P1 recombinants spanning the whn locus [M. Nehls et al., Mamm. Genome 6, 321 (1995)] were partially digested with Sau 3Al, selected by size, and cloned into XKO [M. Nehls, M. Messerle, A. Sirulnik, A. J. H. Smith, T. Boehm, Biotechniques 17, 770 (1994)], which supplies thymidine kinase genes at either end of the insert. Recombinants containing exon 3 were analyzed for inserts in which the Sfi I site was at a reasonable distance from one end of the insert, to facilitate subsequent polymerase chain reaction (PCR) screening. In the clone used here (32), the Sfi I site was located about 1.8 kb downstream of the 5′ end of the homologous sequences and about 4.2 kb upstream of the 3′ end of the homologous sequences. Phage DNA was concatamerized by ligation at its cos ends and then cut with Sfi I. The Sfi I ends were modified by addition of Bam HI adapter oligonucleotides and this DNA subsequently ligated to a Bam HI cassette containing a promoterless β-galactosidase gene preceded by an internal ribosomal entry sequence followed by a neomycin resistance gene (7). The ligation was packaged in vitro, and desired recombinants were selected by plaque hybridization, converted into plasmid form, and linearized with Not I. This linear molecule was then ligated to a self-complementary Not I-compatible, phosphorylated oligonucleotide (5′-GGCCTCCGGTACATGATCGAGGGGACTGACAAGACGGCCAGTCCTCGATCATGTACCGGA-3′) to seal the ends of the linear fragment. This treatment enhances the stability of transfected DNA, improves positive-negative selection, and increases the frequency of homologous recombination (M. Messerle and T. Boehm, in preparation). The targeting construct was transfected into CJ7 cells [P. J. Swiatek and T. Gridley, Genes Dev. 7, 2071 (1993)], and clones resistant to G418 and gancyclovir were analyzed by PCR and Southern (DNA) blotting for homologous recombination. Of 25 colonies tested, 23 were homologous integrants. Mice heterozygous for the insertional mutation were derived as described [A. Warren et al., Cell 15, 45 (1994)].
28. The targeting construct was derived as follows. P1 recombinants spanning the whn locus [M. Nehls et al., Mamm. Genome 6, 321 (1995)] were partially digested with Sau 3Al, selected by size, and cloned into XKO [M. Nehls, M. Messerle, A. Sirulnik, A. J. H. Smith, T. Boehm, Biotechniques 17, 770 (1994)], which supplies thymidine kinase genes at either end of the insert. Recombinants containing exon 3 were analyzed for inserts in which the Sfi I site was at a reasonable distance from one end of the insert, to facilitate subsequent polymerase chain reaction (PCR) screening. In the clone used here (32), the Sfi I site was located about 1.8 kb downstream of the 5′ end of the homologous sequences and about 4.2 kb upstream of the 3′ end of the homologous sequences. Phage DNA was concatamerized by ligation at its cos ends and then cut with Sfi I. The Sfi I ends were modified by addition of Bam HI adapter oligonucleotides and this DNA subsequently ligated to a Bam HI cassette containing a promoterless β-galactosidase gene preceded by an internal ribosomal entry sequence followed by a neomycin resistance gene (7). The ligation was packaged in vitro, and desired recombinants were selected by plaque hybridization, converted into plasmid form, and linearized with Not I. This linear molecule was then ligated to a self-complementary Not I-compatible, phosphorylated oligonucleotide (5′-GGCCTCCGGTACATGATCGAGGGGACTGACAAGACGGCCAGTCCTCGATCATGTACCGGA-3′) to seal the ends of the linear fragment. This treatment enhances the stability of transfected DNA, improves positive-negative selection, and increases the frequency of homologous recombination (M. Messerle and T. Boehm, in preparation). The targeting construct was transfected into CJ7 cells [P. J. Swiatek and T. Gridley, Genes Dev. 7, 2071 (1993)], and clones resistant to G418 and gancyclovir were analyzed by PCR and Southern (DNA) blotting for homologous recombination. Of 25 colonies tested, 23 were homologous integrants. Mice heterozygous for the insertional mutation were derived as described [A. Warren et al., Cell 15, 45 (1994)].
29. The targeting construct was derived as follows. P1 recombinants spanning the whn locus [M. Nehls et al., Mamm. Genome 6, 321 (1995)] were partially digested with Sau 3Al, selected by size, and cloned into XKO [M. Nehls, M. Messerle, A. Sirulnik, A. J. H. Smith, T. Boehm, Biotechniques 17, 770 (1994)], which supplies thymidine kinase genes at either end of the insert. Recombinants containing exon 3 were analyzed for inserts in which the Sfi I site was at a reasonable distance from one end of the insert, to facilitate subsequent polymerase chain reaction (PCR) screening. In the clone used here (32), the Sfi I site was located about 1.8 kb downstream of the 5′ end of the homologous sequences and about 4.2 kb upstream of the 3′ end of the homologous sequences. Phage DNA was concatamerized by ligation at its cos ends and then cut with Sfi I. The Sfi I ends were modified by addition of Bam HI adapter oligonucleotides and this DNA subsequently ligated to a Bam HI cassette containing a promoterless β-galactosidase gene preceded by an internal ribosomal entry sequence followed by a neomycin resistance gene (7). The ligation was packaged in vitro, and desired recombinants were selected by plaque hybridization, converted into plasmid form, and linearized with Not I. This linear molecule was then ligated to a self-complementary Not I-compatible, phosphorylated oligonucleotide (5′-GGCCTCCGGTACATGATCGAGGGGACTGACAAGACGGCCAGTCCTCGATCATGTACCGGA-3′) to seal the ends of the linear fragment. This treatment enhances the stability of transfected DNA, improves positive-negative selection, and increases the frequency of homologous recombination (M. Messerle and T. Boehm, in preparation). The targeting construct was transfected into CJ7 cells [P. J. Swiatek and T. Gridley, Genes Dev. 7, 2071 (1993)], and clones resistant to G418 and gancyclovir were analyzed by PCR and Southern (DNA) blotting for homologous recombination. Of 25 colonies tested, 23 were homologous integrants. Mice heterozygous for the insertional mutation were derived as described [A. Warren et al., Cell 15, 45 (1994)].
30. The targeting construct was derived as follows. P1 recombinants spanning the whn locus [M. Nehls et al., Mamm. Genome 6, 321 (1995)] were partially digested with Sau 3Al, selected by size, and cloned into XKO [M. Nehls, M. Messerle, A. Sirulnik, A. J. H. Smith, T. Boehm, Biotechniques 17, 770 (1994)], which supplies thymidine kinase genes at either end of the insert. Recombinants containing exon 3 were analyzed for inserts in which the Sfi I site was at a reasonable distance from one end of the insert, to facilitate subsequent polymerase chain reaction (PCR) screening. In the clone used here (32), the Sfi I site was located about 1.8 kb downstream of the 5′ end of the homologous sequences and about 4.2 kb upstream of the 3′ end of the homologous sequences. Phage DNA was concatamerized by ligation at its cos ends and then cut with Sfi I. The Sfi I ends were modified by addition of Bam HI adapter oligonucleotides and this DNA subsequently ligated to a Bam HI cassette containing a promoterless β-galactosidase gene preceded by an internal ribosomal entry sequence followed by a neomycin resistance gene (7). The ligation was packaged in vitro, and desired recombinants were selected by plaque hybridization, converted into plasmid form, and linearized with Not I. This linear molecule was then ligated to a self-complementary Not I-compatible, phosphorylated oligonucleotide (5′-GGCCTCCGGTACATGATCGAGGGGACTGACAAGACGGCCAGTCCTCGATCATGTACCGGA-3′) to seal the ends of the linear fragment. This treatment enhances the stability of transfected DNA, improves positive-negative selection, and increases the frequency of homologous recombination (M. Messerle and T. Boehm, in preparation). The targeting construct was transfected into CJ7 cells [P. J. Swiatek and T. Gridley, Genes Dev. 7, 2071 (1993)], and clones resistant to G418 and gancyclovir were analyzed by PCR and Southern (DNA) blotting for homologous recombination. Of 25 colonies tested, 23 were homologous integrants. Mice heterozygous for the insertional mutation were derived as described [A. Warren et al., Cell 15, 45 (1994)].
31. -/- mice, indicating an increase of nonlymphoid cells.
32. R. T. Kubo et al., J. Immunol. 142, 2736 (1989).
33. 2 goat antibody to rat IgG-biotin (Jackson) supplemented with normal goat and rabbit sera at 5% each, and streptavidin-FITC (Boehringer Mannheim, FRG). Stained sections were photographed with an Axiophot microscope (Zeiss) with Fujichrome Provia 400 film.
34. We thank T. H. Rabbitts for advice, I. Schuster, S. Höflinger, G. King, and T. Langford for expert technical help, H. Schrewe for CJ7 cells, and W. van Ewijk for antibodies. These studies were supported by a grant from the Deutsche Forschungsgemeinschaft (Bo 1128/2-2). A.J.H.S. is supported by the Association of International Cancer Research.