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.