[25] In vitro RNA synthesis with SP6 RNA polymerase

Krieg, Paul A.; Melton, D.A.

doi:10.1016/0076-6879(87)55027-3

Cited by 707 publications

(293 citation statements)

References 29 publications

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“…T3 and T7 antisense probes were synthesized and hybridized to RNA samples as described in (Krieg and Melton, 1987). The probe for human tal-1R188G;R189G was derived by linearization of a 625 bp partial cDNA clone with SacI, resulting in a probe that protects 500 nucleotides.…”

Section: Rnase Protection Analysismentioning

confidence: 99%

The DNA binding activity of TAL-1 is not required to induce leukemia/lymphoma in mice

et al. 2001

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Activation of the basic helix ± loop ± helix (bHLH) gene TAL-1 (or SCL) is the most frequent gain-of-function mutation in pediatric T cell acute lymphoblastic leukemia (T-ALL). Similarly, mis-expression of tal-1 in the thymus of transgenic mice results in the development of clonal T cell lymphoblastic leukemia. To determine the mechanism(s) of tal-1-induced leukemogenesis, we created transgenic mice expressing a DNA binding mutant of tal-1. Surprisingly, these mice develop disease, demonstrating that the DNA binding properties of tal-1 are not required to induce leukemia/lymphoma in mice. However, wild type tal-1 and the DNA binding mutant both form stable complexes with E2A proteins. In addition, tal-1 stimulates di erentiation of CD8-single positive thymocytes but inhibits the development of CD4-single positive cells: e ects also observed in E2A-de®cient mice. Our study suggests that the bHLH protein tal-1 contributes to leukemia by interfering with E2A protein function(s). Oncogene (2001) 20, 3897 ± 3905.

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Section: Rnase Protection Analysismentioning

confidence: 99%

The DNA binding activity of TAL-1 is not required to induce leukemia/lymphoma in mice

et al. 2001

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“…RNA from the equivalent of five embryos was then assayed by RNase protection as described by Krieg and Melton (1987) using a 375 nucleotide antisense probe derived from the VEGF coding region. This probe does not distinguish between different alternatively spliced VEGF transcripts.…”

Section: Rnase Protection Analysis Of Flk-1 and Vegf Expressionmentioning

confidence: 99%

Neovascularization of the Xenopus embryo

et al. 1997

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The receptor tyrosine kinase, Flk-1 or VEGFR-2, and its ligand, vascular endothelial growth factor (VEGF) are required for the development of the embryonic vasculature. Targeted disruption of either gene in mice results in the failure of vascular system formation. The Xenopus homologues of flk-1 and VEGF have been cloned and their expression has been examined throughout early embryonic development. These studies indicate that flk-1 is expressed in groups of endothelial precursor cells which will form the major blood vessels of the embryo, including the posterior cardinal veins, the dorsal aorta, the vitelline veins, and the endocardium. VEGF expression is found in tissues adjacent to the mesenchyme containing the flk-1-expressing endothelial precursors. Expression of both flk-1 and VEGF is transient, appearing as the primary vascular plexus is forming and declining steadily after the onset of functional embryonic circulation. After establishment of the primary vascular structures, flk-1 expression is also observed in the intersegmental veins which form by an angiogenic mechanism. Overall, these results support a role for VEGF/flk-1 signaling in both vasculogenesis and angiogenesis in the Xenopus embryo. When VEGF is expressed ectopically in Xenopus embryos by microinjection of either plasmid DNA or synthetic mRNA, large, disorganized vascular structures are produced. This result indicates that ectopic VEGF is capable of altering the architecture of the developing vascular network.

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“…For the production of capped transcripts, the analog m 7 G(5 0 )ppp(5 0 )G was included in the reaction mixture at 1 mM concentration. 29,61 All the reagents were supplied from Promega (Madison, WI, USA). For the transfection experiments, the transcription mixtures were purified by gel filtration using the Quick Spin Columns kit for RNA purification (Roche Diagnostics, Indianapolis, IN, USA).…”

Section: In Vitro Transcription and Transfectionmentioning

confidence: 99%