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DNA Chips

DNA Chips. Attach DNA to tiny spots on glass slides (i.e., chip). Hybridize fluorescently-labeled DNA probes to chip. Detect hybridization to different probes with chip reader. Two different kinds of DNA chips. cDNA chips:

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DNA Chips

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  1. DNA Chips • Attach DNA to tiny spots on glass slides (i.e., chip). • Hybridize fluorescently-labeled DNA probes to chip. • Detect hybridization to different probes with chip reader. Two different kinds of DNA chips cDNA chips: Machine puts many tiny spots of different cDNAs onto glass slide. Oligonucleotide chips: Synthesize thousands of different oligos in spots on chip. Each oligo may have part of the sequence of a different gene in the genome.

  2. Synthesis of an oligonucleotide chip

  3. Synthesize fluorescently-labeled cDNA probes using mRNA from different kinds of cells. Hybridize this mixed cDNA probe to chips. Intensity of hybridization signals indicate relative levels of respective mRNAs. Top: cDNA chip hybridized to cDNA probe made from bone marrow mRNA. Bottom: Oligonucleotide array chip hybridized with tissue- specific cDNA probe.

  4. cyc = cycloheximide Examine changes in expression of many genes under different conditions Rows = different genes Columns = different time points Another example: Compare expression of genes in normal cells to that in various kinds of cancer cells. Red = mRNA levels above control Green = mRNA levels below control

  5. Transgenesis Putting cloned gene into genome of animal or plant • Two approaches for making transgenic mice: • Ectopic insertion: • Introduce DNA at random locations. • Gene Replacement: • Replace part or all of endogenous gene with cloned version of gene by homologous recombination. • Can replace mutant allele with wild type allele, or replace wild type allele with mutant allele.

  6. Ectopic Insertion Inject DNA into nucleus of single-cell mouse embryo. DNA integrates at random location as tandem arrays. Alternatively, use retrovirus vector to integrate DNA as single copy at random locations.

  7. Gene Replacement Making knock-out mutation Kills cells that don’t have neor gene Double cross- over Kills cells that have tk gene neor tk neor Embryonic stem cells

  8. Embryonic Stem (ES) Cells Produced from cells within early embryos that are pluripotent. Pluripotent = can develop into ~any kind of cells in body. ES cells can be grown indefinitely in culture & are relatively easy to genetically modify.

  9. Making Transgenic Mice Cell containing targeted mutation

  10. Breed chimeric mice After two addition generations, can produce homozygotes

  11. Summary: Making Transgenic Mice • Make targeted knock-out mutation of specific gene in ES cells. • Inject genetically modified ES cells into blastocyst- stage embryos & implant in surrogate mother. • Resulting adult mice should be somatic chimeras & some should also be germ line chimeras. • Do genetic crosses & use PCR to screen for progeny that are heterozygous for the targeted mutation. • Cross heterozygous mice to each other & examine phenotypes of homozygous progeny.

  12. Gene Therapy Clone the gene that is responsible for a genetic disease. Put a wild type copy of gene into either the germ line or somatic cells of patient. Germ line therapy: - Introduce DNA into embryonic stem cell and inject genetically modified ES cell into early embryo. - Some cells of adult would contain the transgene, others would not. - If some of the genetically modified cells make up at least part of the germ cell population, then this genetic modification would be passed on to the next generation. Somatic therapy: - Remove some of patient’s somatic cells. - Put a wild type copy of gene into those cells & reintroduce those cells into the patient. - Hope that wild type transgene is expressed correctly & wild type protein,which is missing in the mutant cells, is produced.

  13. Gene Therapy

  14. Viral vectors Defective viruses used as vectors -- lack viral genes needed for replication. Retrovirus vectors: -Retroviruses have RNA genomes, that, once inside cell, are reverse transcribed into DNA & this DNA copy is integrated into host cell’s genome. - Integrated retrovirus’ genes transcribed & replicated like other chromosomal genes. - By inserting a cloned gene into a defective retrovirus vector, can get a cloned gene integrated into human cell’s genome, but can’t produce more viruses. Ex: Has been successfully used to treat SCID. SCID caused by mutation in ADA gene, which prevents production of antibodies. Problem: Some patients have developed leukemia, caused by insertion of retrovirus. Adenovirus vectors: DNA virus that infects lung cells. May be able to use defective adenovirus vectors to treat respiratory diseases, such as cystic fibrosis.

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