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 tissue-specific cDNA probe.

Red = mRNA levels above control

Green = mRNA levels below control

cyc = cycloheximide

Examine changes in expression of many genes under different conditions

Another example:Compare expression of genes in normal cells to that in various kinds of cancer cells.

Rows = different genes

Columns = different time points

TransgenesisPutting 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 InsertionInject 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

Embryonic stem cells

Making knock-out mutation

Kills cells that have tk gene

Kills cells that don’t have neor gene

tk

neor

neor

Double cross-over

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 miceAfter two addition generations, can produce homozygotes

• 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.

Summary: Making Transgenic Mice

Gene Therapy1. 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

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.

Defective viruses used as vectors -- lack viral genes needed for replication.