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US4981790A Expired / Lapsed US ⏱️ 0.0 years remaining

Stable TatIII cell lines, TatIII gene products, and assay methods

Assignee: Dana Farber Cancer Institute Inc
•
Filing: 1985-12-06
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Granted: 1991-01-01
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Expires: 2005-12-06
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Abstract

Abstract This invention describes stable tatIII cell lines. It is disclosed that by transfecting a preselected tatIII cell line with a vector containing a sufficient amount of the HTLV-III LTR responsive to tatIII gene products for trans-activation and an enhancer upstream of the tatIII responsive segment, it is possible to express high levels of the tatIII gene products. By including a preselected heterologous gene on this vector, it is also possible to express high levels of a desired gene product. A substantially pure protein comprising 86 amino acids and having an apparent molecular weight of about 14,000 dalton and exhibiting trans-activating activity is also disclosed. This protein and polypeptides having trans-activating ability, which is also disclosed, can be used to produce high levels of a desired gene product. A method of detecting the presence of HTLV-III/LAV virus in an individual is also disclosed and comprises the step of: (a) incubating whole blood or lymphocytes from the invididual to be tested with tatIII cell lines of the present invention in a culture medium; and (b) screening for cytopathic effects on the cells is also disclosed. A method of screening for a compound that inhibits trans-activation of the tatIII gene product is also disclosed and comprises the steps of: (1) transacting a tatIII cell line of the present invention with a vector containing a gene that expresses a selectable marker and whose expression is under the control of an HTLV-III LTR; (2) transfecting the same type of tatIII cell lines as in step (1) with the selectable gene chosen in step (1) but under the control of a different regulatory sequence; (3) thereafter, adding a preselected compound to each of the cell lines in increasing concentrations; and (4) measuring the expression of the selectable gene product to determine whether the compound effects the tatIII function without being toxic to the cell.

Inventors

  • 👤 William A. Haseltine
  • 👤 Craig A. Rosen
  • 👤 Joseph G. Sodroski
  • 👤 Wei C. Goh

Bibliographic Details

Application Number —
Jurisdiction US
Data Source google_patents
Last Synchronized 2026-11-04 10:02
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/005; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C12N2740/00; C12N2740/00011; C12N2740/10011; C12N2740/14011; C12N2740/14022; C12N2740/16011; C12N2740/16311; C12N2740/16322

Claims Analysis

Total Claims: 19
Claim 1
Independent Claim 97 words
A method for enhanced expression of heterologous DNA comprising: (a) transfecting a cell with (1) a vector containing a sufficient portion of an HTLV-III tat III gene to produce a tat III gene product which acts as a trans-activator on an HTLV-III LTR or fragment thereof, (2) a second vector containing a sufficient amount of an HTLV-III LTR which is capable of responding to the tat III gene product, and an enhancer upstream of said LTR or functional fragment thereof and the heterologous DNA downstream of the LTR or fragment thereof; and (b) culturing the co-transferred cell.
Claim 2
Independent Claim 97 words
A method for enhanced expression of heterologous DNA comprising: (a) transfecting a cell with (1) a vector containing a sufficient portion of an HTLV-III tat III gene to produce a tat III gene product which acts as a trans-activator on an HTLV-III LTR or fragment thereof, (2) a second vector containing a sufficient amount of an HTLV-III LTR which is capable of responding to the tat III gene product, and an enhancer upstream of said LTR or functional fragment thereof and the heterologous DNA downstream of the LTR or fragment thereof; and (b) culturing the co-transferred cell.
Claim 3
Dependent Claim 14 words
The method of claim 1, wherein the product of the heterologous DNA is recovered.
Claim 4
Dependent Claim 16 words
The method of claim 1, wherein the LTR is the 5' LTR or functional fragment thereof.
Claim 5
Independent Claim 40 words
A method for producing a stable cell line containing the tat III gene which comprises: (a) transfecting a preselected cell with a vector containing a tat III gene but not the complete HTLV-III/LAV genome, and (b) culturing the transfected cells.
Claim 6
Independent Claim 40 words
A method for producing a stable cell line containing the tat III gene which comprises: (a) transfecting a preselected cell with a vector containing a tat III gene but not the complete HTLV-III/LAV genome, and (b) culturing the transfected cells.
Claim 7
Dependent Claim 15 words
A stable cell line containing the tat III gene, but not the complete HTLV-III/LAV genome.
Claim 8
Dependent Claim 15 words
A stable cell line containing the tat III gene, but not the complete HTLV-III/LAV genome.
Claim 9
Dependent Claim 21 words
The method of claim 1 wherein the sufficient amount is at least from nucleotides -17 through +80 of the HTLV-III LTR.
Claim 10
Dependent Claim 14 words
The method of claim 1, wherein the sufficient amount is the HTLV-III TAR element.
Claim 11
Dependent Claim 21 words
The method of claim 1 wherein the sufficient amount is at least from nucleotides -167 through +80 of the HTLV-III LTR.
Claim 12
Dependent Claim 15 words
The method of claim 8 wherein the viral enhancer is part of the HTLV-III LTR.
Claim 13
Dependent Claim 16 words
The method of claim 1 wherein the heterologous DNA is the gene coding for chloraphenicol acetyltransferase.
Claim 14
Dependent Claim 17 words
The tat III cell line of claim 5 wherein the cell line is a Raji cell line.
Claim 15
Dependent Claim 17 words
The tat III cell line of claim 5 where the cell line is a HeLa cell line.
Claim 16
Dependent Claim 19 words
The tat III cell line of claim 5 where the cell line is a NIH 3T 3 cell line.
Claim 17
Dependent Claim 17 words
The tat III cell line of claim 5 where the cell line is a Jurkat cell line.
Claim 18
Dependent Claim 17 words
The tat III cell line of claim 5 where the cell line is a human T-cell line.
Claim 19
Dependent Claim 17 words
The tat III cell line of claim 5 where the cell line is a human B-cell line.

References Cited (Backward Citations)

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USPTO Annuity & Maintenance Windows

Annuity Stage Window Opens Payment Deadline Surcharge Period Until Estimated Fee Status
3.5 Years (Year 4) 1993-12-31 1994-07-02 1995-01-01 $2000.00 Window Closed / Lapsed
7.5 Years (Year 8) 1997-12-31 1998-07-02 1999-01-01 $3760.00 Window Closed / Lapsed
11.5 Years (Year 12) 2001-12-31 2002-07-02 2003-01-01 $7700.00 Window Closed / Lapsed

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