Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1998-10-21
2000-11-28
Myers, Carla J.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, 435 9151, 536 2431, 536 2433, C12Q 168, C12P 1934, C07H 2104
Patent
active
061533858
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a process for detecting the expression of CD95 ligand in cells.
BACKGROUND OF THE INVENTION
Apoptosis is the designation for programmed cell death. It is found e.g. in organogenesis and metamorphosis, tissue atrophy and tumor regression. Apoptosis is linked with a condensation of the cytoplasm, a loss of plasma membrane villi, a segmentation of the nucleus and extensive degradation of chromosomal DNA. It has turned out that disturbances of apoptosis occur in various diseases such as AIDS, autoimmune diseases, tumors and leukemias. In the case of AIDS, increased apoptosis seems to be responsible for the strong decrease of the CD4-T cells.
In activated T cells and in other cells there is found a cell surface protein referred to as CD95 and APO-1, respectively. A soluble or membrane-bound protein referred to as CD95 ligand and APO-1 ligand, respectively, can bind to this cell surface protein and trigger the induction of apoptosis.
The extent and course of apoptosis as well as the molecules causing the same cannot be determined so far. However, this would be necessary to be able to carry out e.g. follow-ups and suitable therapeutic measures in patients suffering from the above-mentioned diseases.
Therefore, it is the object of the present invention to provide a product serving for determining the extent of apoptosis and its course, respectively.
SUMMARY OF THE INVENTION
The present invention relates to a process for quantitatively determining CD95 ligand, comprising the following processing steps: transcription, and fragment by CD95 ligand-specific primers in a PCR reaction.
The process is suitable for determining the extent and/or course of apoptosis.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a diagram of the quantitative PCR according to the invention for CD95-L.
FIG. 2 shows a diagram for the preparation of a competitor DNA fragment of CD95-L. L.sub.up and L.sub.down, are shown. in the middle of the sequence and contains an overhanging sequence at the 5' end having a length of 50 bp. at the 5' end of L.sub.downOV are overhanging and complementary to the last 30 bp at the 5' end of L.sub.upOV. The complementary regions are shown in brush-shaped fashion. long DNA pieces. Following purification, both fragments are hybridized with each other via their complementary regions. reaction where L.sub.up and L.sub.down are used again as primers. insert in the middle of the sequence.
FIG. 3 shows a constitutive and induced expression of CD95-L mRNA in lymphoid cells.
DETAILED DESCRIPTION OF THE INVENTION
It is the object of the present invention to provide a product serving for determining the extent of apoptosis and its course. According to the invention this is achieved by the subject matters defined in the claims.
Therefore, the subject matter of the present invention relates to a process by which the CD95 ligand, hereinafter referred to as CD95-L, can be determined quantitatively in cells.
The present invention is based on the applicant's finding that apoptosis is caused in T cells by an increased amount of CD95-L. Furthermore, the applicant discovered that the apoptosis rate is determined by the CD95-L amount.
The process according to the invention relates to a quantitative "polymerase chain reaction" (PCR) by which the amount of CD95-L in cells can be determined reliably. The amount of CD95-L mRNA present in the cell is determined in the process according to the invention by an RT-PCR reaction. In the quantitative PCR for CD95-L, the amount of CD95-L cDNA is titrated by adding a CD95-L-specific DNA competitor fragment to the PCR reaction. The complete reverse transcription of a defined amount of total RNA into cDNA precedes this step. The competitor fragment differs from the "wild-type" cDNA of CD95-L by additional base pairs in the middle of the nucleotide sequence but has identical flanking sequences. Therefore, wild-type and competitor fragment are recognized by the same primer combination and can be multiplied jointly in a PCR. Due to t
REFERENCES:
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Chomczynski and Sacchi, 1987, Anal. Biochem. 162:156-159.
Debatin et al., 1995, "Involvement of the APO-1 (FAS-CD95) system in T cell depletion in AIDS," Blood 86(10 Suppl. 1):288A; 37th Annual Meeting of American Society of Hematology, Seattle, Washington, USA Dec. 1-5, 1995.
Dhein et al., 1995, "Autocrine T-cell suicide by APO-1," Nature 373:438-441.
Herr et al., 1995, "Development of a quantitative RT-PCR for the human APO-1 (FAS-CD95) ligand and monitoring of ligand expression in normal and malignant Tcells," Blood 86 (10 Suppl. 1):163A; 37th Annual Meeting of the American Society of Hematology, Seattle, Washington, USA, Dec. 1-5, 1995.
Herr et al., 1993, "Monitoring of CD95 ligand expression in human T cells by quantitative RT-PCR," Cell Death and Differentiation 3:299-305.
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Debatin Klaus-Michael
Herr Ingrid
Deutsches Krebsforschungszentrum Stiftung des Offentlichen Recht
Johannsen Diana
Myers Carla J.
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