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S, A, Nedospasov el at, ibid, 14, 7713 (1986
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Ibid
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B. S. Conta el a!., ibid 134. 2185 (1985
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Ibid
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Conta, B.S.1
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L. A. Tartaglia et al, Ceil 73, 213 (1993)
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J. H. Kehrl el al., ibid 238, 1144 (1987
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Ibid
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Kehrl, J.H.1
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0023233959
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The genes encoding LT and TNF-A were isolated on a single clone irom a BALB/c cosmtd library
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V, C. Broudy, J. M. Harlan, J W, Adamson, J Immunol 138. 4298 (1987). The genes encoding LT and TNF-A were isolated on a single clone irom a BALB/c cosmtd library
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J Immunol
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Broudy, V.C.1
Harlan, J.M.2
Adamson, J.W.3
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21
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84995838286
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with pMuCachectin complementary DNA (cDNA) as the probe. A 4,5-kb Hind Ill-Xba I fragment containing the gene encoding LT was subcloned into pBJuescript KS(+) (Strat-Agene) containing the gene encoding herpes simplex virus-thymidine kinase (HSV-tk) under the control of a polyoma promoter and enhancer
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The genes encoding LT and TNF-A were isolated on a single clone irom a BALB/c cosmtd library |D. D. Chaplin elal., Proc. Natl. Acad. So. U.S.A. 80, 6947 (1983)], with pMuCachectin complementary DNA (cDNA) as the probe. A 4,5-kb Hind Ill-Xba I fragment containing the gene encoding LT was subcloned into pBJuescript KS(+) (Strat-Agene) containing the gene encoding herpes simplex virus-thymidine kinase (HSV-tk) under the control of a polyoma promoter and enhancer
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(1983)
Proc. Natl. Acad. So. U.S.A.
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Chaplin, D.D.1
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22
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0024296027
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A 420-bp Apa I-Kpn I tragment containing three nucieotides of LT exon 2 through the first five nucleotides of LT exon 4 was replaced by a linker containing a Sal I site. A 1.6-kb Xho I fragment encoding neornycin resistance {neo under the control of the pgk promoter
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S. L Mansour, K. R. Thomas, M. R. Capecchi. Nature 336, 348 (1988)]. A 420-bp Apa I-Kpn I tragment containing three nucieotides of LT exon 2 through the first five nucleotides of LT exon 4 was replaced by a linker containing a Sal I site. A 1.6-kb Xho I fragment encoding neornycin resistance {neo under the control of the pgk promoter
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(1988)
Nature
, vol.336
, pp. 348
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Mansour, S.L.1
Thomas, K.R.2
Capecchi, M.R.3
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23
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0026023289
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The linearized vector was electroporated into the D3 ES line, and cells were plated on primary embryonic fibroblasts from day 14 embryos obtained by mating a male C1D mouse (GenPharm, International. Mountain View. CA) with a female Swiss Webster mouse (Taconic Farms, Germantown, NY). Selection was with 2 iiWi ganciclovir (Cytovene, Syntex Laboratories, Palo Alto, CA) and G418 (250 ng/ml
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P. Soriano, C. Montgomery, R Geske, A Bradley, Cel/64, 693 (1991)] was inserted into the Sal I site in the opposite transcriptional orientation compared to the gene encoding LT. The plasmid contains 2.5 kb that is homologous to LT genomic DNA 5' of the neo' gene and 0,88 kb (also homologous to LT genomic DNA) 3' of the neor gene. It predicts a transcript from the LT promoter that contains the LT 5' untranslated sequence and most of the LT signal peptide [93 nucleotides (nt) in exon 2], followed by 1660 nt of the reverse orientation neo' fragment, 986 nt of the LT exon 4 and a polyadenylate (poly(A)] tail [total anticipated size is 2901 nt plus the poly(A) tail]. A termination codon is located 276 nt into the antisense neor fragment. Thus, the transcript predicts a polypeptide product of 31 residues of the LT signal peptide followed by 91 residues ot an-lisense neo' sequence only. The linearized vector was electroporated into the D3 ES line, and cells were plated on primary embryonic fibroblasts from day 14 embryos obtained by mating a male C1D mouse (GenPharm, International. Mountain View. CA) with a female Swiss Webster mouse (Taconic Farms, Germantown, NY). Selection was with 2 iiWi ganciclovir (Cytovene, Syntex Laboratories, Palo Alto, CA) and G418 (250 ng/ml; Gibco-BRL). Double drug-resistant colonies were screened by Southern blotting with an LT-TNF-A iniergenic probe Homologous recombination was detected at a frequency of 1 in 63 drug-resistant colonies Chimeric animals were obtained by injecting targeted cells into CS7BL/6 blastocysts. Male mice showing <50% coat color chimerism were mated to C57BL76 females, and germline transmission was documented by the presence of the 5 -kb Pst I fragment
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(1991)
Cel
, vol.64
, pp. 693
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Soriano, P.1
Montgomery, C.2
Geske, R.3
Bradley, A.4
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25
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0025000863
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Bioaciivity of TNF-A-LT was measured with the WEH1164 librosarcoma cell line as described The specificity of fhe assay was defined with a neutralizing monoclonal antibody, TN3 19 12. that recognizes both murine TNF-A and LT.
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Bioaciivity of TNF-A-LT was measured with the WEH1164 librosarcoma cell line as described [N. H. Ruddle etal. J. Exp. Med. 172. 1193 (1990)]. The specificity of fhe assay was defined with a neutralizing monoclonal antibody, TN3 19 12. that recognizes both murine TNF-A and LT.
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(1990)
J. Exp. Med.
, vol.172
, pp. 1193
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Ruddle, N.H.1
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26
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84896514132
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Erythrocyie-depleted cells (106) were stained for 1 hour with fluoresceinated monoclonal antibodies to CDS (Beoton Dickinson), phycoeryihrin-labeled monoclonal antibodies to CD4 (Becton Dickinson), fluoresceinated monoclonal antibodies to IgM (Pharmingen). phycoerythrin-labeled monoclonal antibodies to CD45R and B220 (Pharmingen) h or biotmylated monoclonal antibodies to CD3e (Pharmingen). The biotmylated monoclonal antibody was detected with strepta-vidin-RED613 (Gibco). Flow cytometnc analysis was performed with the Lysis-I program (Becton Dickinson) on a FACScan (Becton Dickinson); data are means from experiments with three animals
-
Erythrocyie-depleted cells (106) were stained for 1 hour with fluoresceinated monoclonal antibodies to CDS (Beoton Dickinson), phycoeryihrin-labeled monoclonal antibodies to CD4 (Becton Dickinson), fluoresceinated monoclonal antibodies to IgM (Pharmingen). phycoerythrin-labeled monoclonal antibodies to CD45R and B220 (Pharmingen) h or biotmylated monoclonal antibodies to CD3e (Pharmingen). The biotmylated monoclonal antibody was detected with strepta-vidin-RED613 (Gibco). Flow cytometnc analysis was performed with the Lysis-I program (Becton Dickinson) on a FACScan (Becton Dickinson); data are means from experiments with three animals,
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27
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84896523625
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Cytolytic effector cells were established by primary, one-way mixed lymphocyte cultures of 1 x 10' to 2 x 10'LT-or LT-'-splenocytes (H-2) with 2x10' irradiated [absorbed dose of radiation (Gy) = 20 Gy] BALB/C [H-2splenocytes. Responding cells were either unfractionated splenocytes. purified T cells (eluled from nylon wool then treated with monoclonal antibody J11 d and complement), or purified CD4T cells (eluted from nylon wool then treated with J11d and the CDB monoclonal antibody 3155 and complement). After culture for 5 to 6 days, live effector cells were isolated by Ficoll-Hypaque density gradient centrifugation. Target cells (0.5 x 10e to 10 x 106) were labeled in 1 ml of RPMI-1640for 90 min with 500 |j.Ci of Na/'CrO, or 10 (iCi of 5r '-labeled iododeoxyuridine (15s[-UdR) as indicated, washed, incubated in the absence of label for 30 to 45 min. washed again, and added to varying numbers of effector cells
-
Cytolytic effector cells were established by primary, one-way mixed lymphocyte cultures of 1 x 10' to 2 x 10'LT-or LT-'-splenocytes (H-2) with 2x10' irradiated [absorbed dose of radiation (Gy) = 20 Gy] BALB/C [H-2splenocytes. Responding cells were either unfractionated splenocytes. purified T cells (eluled from nylon wool then treated with monoclonal antibody J11 d and complement), or purified CD4T cells (eluted from nylon wool then treated with J11d and the CDB monoclonal antibody 3155 and complement). After culture for 5 to 6 days, live effector cells were isolated by Ficoll-Hypaque density gradient centrifugation. Target cells (0.5 x 10e to 10 x 106) were labeled in 1 ml of RPMI-1640for 90 min with 500 |j.Ci of Na/'CrO, or 10 (iCi of 5r '-labeled iododeoxyuridine (15s[-UdR) as indicated, washed, incubated in the absence of label for 30 to 45 min. washed again, and added to varying numbers of effector cells. At the indicated times, release of label into the medium was assessed. Alter 6 hours, spontaneous 51Cr release of P815 cells (H-2) was 7%, of TA3 cells (H-S) was 21%, and of EL-4 cells {H-2) was S%. The nuclear lesion was assessed by termination of the incubation with 1 mM EDTA and 0,2% Triton X-100. Spontaneous release of la5lUdR was 14%.
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31
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0024564252
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M. L Casey, S. M. Cox, B, Beutler. L. Milewich. P. C. MacDonald. J. Clin. Invest 83, 430 (1989)
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S. Miyawaki etal., Eur. J. Immunol. 24, 429 (1994) 37 We gratefully acknowledge P. M Allen. L E.
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Miyawaki, S.1
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39
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84896508196
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for helpful discussions concerning murine lymphoid development and lymph node and spleen architecture; O. Kanagawa tor discussion of a/ymice, C. Bergman for LT-TNF bioassays, B. Beutler at the University of Texas, Southwestern Medical School, for pMuCachectin cDNA, T. Doetschrnan at the Universisy of Cincinnati for the D3 ES cell ine, and E. R. Unanue far evaluation ol murine histcpathclogy. Supported in part by NIH grants CA 16885 (N.H.R) and CA 28533 (J.H.R.), and
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Fields. M. Peters, and E. R. Unanue for helpful discussions concerning murine lymphoid development and lymph node and spleen architecture; O. Kanagawa tor discussion of a/ymice, C. Bergman for LT-TNF bioassays, B. Beutler at the University of Texas, Southwestern Medical School, for pMuCachectin cDNA, T. Doetschrnan at the Universisy of Cincinnati for the D3 ES cell ine, and E. R. Unanue far evaluation ol murine histcpathclogy. Supported in part by NIH grants CA 16885 (N.H.R) and CA 28533 (J.H.R.), and
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Peters, F.M.1
Unanue, E.R.2
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40
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84896499565
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National Multiple Sclerosis Society grant RG-2394 (N.H.R.). D.D.C. is an investigator of the Howard Hughes Medical Institute 17 December 1993; accepted 11 March 1994
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National Multiple Sclerosis Society grant RG-2394 (N.H.R.). D.D.C. is an investigator of the Howard Hughes Medical Institute 17 December 1993; accepted 11 March 1994
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