Transcript 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: 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. Synthesis of an oligonucleotide chip 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 tissuespecific cDNA probe. 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 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. 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. Gene Replacement Making knock-out mutation Double crossover neor tk neor Embryonic stem cells Kills cells that don’t have neor gene Kills cells that have tk gene 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. Making Transgenic Mice Cell containing targeted mutation Breed chimeric mice After two addition generations, can produce homozygotes Summary: Making Transgenic Mice • Make targeted knock-out mutation of specific gene in ES cells. • Inject genetically modified ES cells into blastocyststage 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. Gene Therapy 1. Clone the gene that is responsible for a genetic disease. 2. 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. Gene Therapy 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.