Novel gene having reverse transcriptas motif
a technology of reverse transcriptase and gene, applied in the field of new gene having a reverse transcriptase motif, can solve the problems of unfavorable and effective methods for measuring telomerase activity, and much is yet unknown about telomeras
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Benefits of technology
Problems solved by technology
Method used
Image
Examples
application examples
[0178] As generally explained above, gene therapy and various other applications can be achieved by using the whole or part of the gene of the invention, the protein encoded thereby, and their mutants. Some examples of these applications will be explained in detail below.
(1) Example of Application to Gene Therapy
[0179] By expressing the antisense RNA of the invention intracellularly, translation into CRT-1 protein can be inhibited.
[0180] For this purpose, the total length of pCRT-1 or part of cDNA is incorporated into a cloning site of an expression vector for a suitable animal cell in the reverse direction, whereby antisense RNA can be prepared with the use of a suitable promotor.
[0181] The above material of the invention is also applicable as an intracellular antibody which suppresses the activity of pCRT-1 protein.
[0182] Concrete examples are HIV therapy (Marasco W. A., Gene Therapy (1997) 4, 11-15) and breast cancer therapy (wright M., et al., Gene Therapy (1997) 4, 317-32...
example 1
(1) Preparation of HL-60 mRNA and Construction of cDNA Library
[0197] Total RNA was prepared from HL-60 strain in accordance with the method of Chirgwin et al. (1979) (Biochemistry 18, 5294-5299), and poly A+ RNA was prepared with the use of mRNA Purification Kit (Pharmacia) in accordance with the manufacturer's instructions.
[0198] HL-60 cDNA libraries for 5′RACE and 3′RACE were constructed with the use of Marathon™ cDNA Amplification Kit (CLONTECH) in accordance with the manufacturer's instructions.
(2) 5′RACE Method
[0199] Primers for amplifying the 5′ region of a gene having a reverse transcriptase-like motif, i.e., hTRT5 (sequence: 5′-ccgctcgtagttgagcacgctgaa-3′) and telo-rev (sequence: 5′-accctcttcaagtgctgtc-3′) were designed based on a human-derived EST clone (GenBank Accession Number AA281296) having high homology to a telomerase catalytic subunit of Ciliophora Euplotes or the yeast S. cerevisiae.
[0200] These primers were obtained from Sawady Technology (as ...
example 2
Genetic Diagnosis by RT-PCR Method
1. Analysis of Expression Distribution
[0226] Using primers (hRT3 and hTRT5) which amplify the gene CRT-1 having a reverse transcriptase-motif according to the invention, the expression of hCRT in cancer cell strains and testis having telomerase activity, and a normal diploid cell strain WI-38 having no telomerase activity was investigated by RT-PCR.
[0227] RT-PCR was performed by the following procedure:
[0228] Total RNA was prepared from various cultured cells in accordance with the guanidine isothianate / phenol method (Chomc zynski and Sacchi (1987) Anal. Biochem. 162,156-159), and cDNA was synthesized with the use of First-Strand cDNA Synthesis Kit (Pharmacia) in accordance with the attached instruction manual.
[0229] Using 1 / 50 of the reaction product as template, PCR was performed under the following conditions:
[0230] The reaction was to be carried out on a 20 μl scale, and a reaction mixture was prepared to comprise 10 mM Tris-HCl, pH 8.3,...
PUM
| Property | Measurement | Unit |
|---|---|---|
| molecular weight | aaaaa | aaaaa |
| pH | aaaaa | aaaaa |
| length | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More