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FROM GENE TO DNA TECHNOLOGY

FROM GENE TO DNA TECHNOLOGY. Case Study: Familial HYPERCHOLESTEROLEMIA. RFLP gene mapping cDNA PCR in-situ hybridization gene cloning “knock-out” genes gene therapy. References: The information about “FH” on these slides is excerpted from Online Mendelian Inheritance in Man

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FROM GENE TO DNA TECHNOLOGY

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  1. FROM GENE TO DNA TECHNOLOGY Case Study: Familial HYPERCHOLESTEROLEMIA RFLP gene mapping cDNA PCR in-situ hybridization gene cloning “knock-out” genes gene therapy References: The information about “FH” on these slides is excerpted from Online Mendelian Inheritance in Man http://www.ncbi.nlm.nih.gov/Omim/

  2. Familial HYPERCHOLESTEROLEMIA Background an autosomal dominant disorder characterized by elevation of serum cholesterol bound to low-density lipoprotein (LDL), resulting in markedly increased cholesterol level and an increased incidence of early onset of atherosclerosis and its complications. Mutations in the LDL receptor (LDLR) gene on chromosome 19 cause this disorder. Occurance: about 7 out of 1000 people.

  3. The LDL receptor was discovered through an extraordinary collaboration between two extraordinary scientists, Michael S. Brown and Joseph L. Goldstein.

  4. Cells that need cholesterol synthesize LDL receptors. from Alberts et al. Molecular Biology of the Cell, Garland Publishing, Third edition, 1994.

  5. Clathrin Pits

  6. Mutations in the LDL receptor (LDLR) gene Horsthemke et al. (1987) suggested that unequal crossing-over between 2 Alu-repetitive DNA sequences was responsible for an intragenic deletion of the LDLR gene leading to familial hypercholesterolemia……..thus supporting a notion of recombination hotspots which involve Alu sequences. The deletion was presumably caused by an unequal crossover event between 2 homologous chromosomes at meiosis. A mutant LDL receptor with ARG 158 replaced by CYS.

  7. A mutant LDL receptor with ARG 158 replaced by CYS. How was DNA technology used to: Locate the LDL receptor (LDLR) gene? Identify individuals who carry LDLR mutations? Study the disease using an animal model? Develop an ex vivo genetic therapy for this disease?

  8. Gene Mapping The LDLRgenewas regionalized to 19p13.1-p13.3 by in situ hybridization (Lindgren et al., 1985). Judging by the sequence of loci suggested by linkage data (pter--FHC--C3--APOE/APOC2), the location of FHC (LDLR) is probably 19p13.2-p13.12 and of C3, 19p13.2-p13.11. Humphries et al. (1985) found a RFLPof the LDL receptor gene using the restriction enzyme PvuII.

  9. Diagnosis Li et al. (1988) worked out a PCR method for analyzing DNA sequences in individual diploid cells and human sperm. Recent progress in PCR technology and and mutation detection methodology have facilitated the rapid screening process for detecting these point mutations among a high number of patients.

  10. …….....The accumulation of cholesterol in patients suffering from FH was suspected to be caused by a defect in the protein LDL-receptor the main function of which was known to lie in the mopping up cholesterol from the circulating blood. In the next step the LDL-receptor gene was cloned from a unaffected individual using its affinity for cholesterol as screening procedure. The final proof for an implication of this protein in the pathogenesis in FH sufferers came from the demonstration that the LDL-receptor gene was mutated in FH-sufferers……………..

  11. Constructing a “Knock-out Mouse DNA Construct Mouse LDL (low density lipoprotein) receptor cDNA was amplified by PCR from mouse liver first strand cDNA using polyA+ RNA and the following primers: Primer A (5'-ATTCT....

  12. Low density lipoprotein receptor Normal fertility and normal triglyceride concentrations. Increased susceptibility to gross atheromata and to thickening of aortic valve leaflets. Increased susceptibility to increased plasma VLDL, IDL, and LDL cholesterol and total plasma cholesterol concentration. Increased susceptibility to develop massive xanthomatousinfiltration of skin and subcutaneous tissues

  13. Wilson et al. (1992) presented a detailed clinical protocol for the ex vivo gene therapy of familial hypercholesterolemia. Their approach involves recovery of hepatocytes from the patient and reimplanting them after genetic correction by a retrovirus-mediated gene transfer. Not only were the technical details of vectors and viruses, transduction and delivery of hepatocytes, evaluation of engraftment and rejection, etc., discussed, but also assessment of risks versus benefits. Gene Therapy

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