transgenic and knockout mice
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Transgenic and knockout mice. Dr. Sumbul Fatma. Mice - Models of Human Diseases. Although the human is the mammal we are generally most interested in learning more about, it is also the one animal we cannot use for genetic experiments for obvious ethical reasons - PowerPoint PPT PresentationTRANSCRIPT
Dr. Sumbul Fatma
Mice - Models of Human DiseasesAlthough the human is the mammal we are generally most
interested in learning more about, it is also the one animal we cannot use for genetic experiments for obvious ethical reasons
Mice naturally develop conditions that mimic human disease, such as cardiovascular disease, cancer and diabetes
Mouse are a favorite model for human disease because it has a relatively low cost of maintenance and a generation time that measures only nine weeks
Developments in molecular biology and stem cell biology have allowed researchers to create custom-made mice through gene targeting in mouse embryonic stem (ES) cells
Certain diseases that afflict only humans, such as cystic fibrosis and Alzheimer's can also be induced by manipulating the mouse genome and environment
ES Cells and Chimeric MiceEmbryonic stem (ES) cells are
pluripotent cell lines with the capacity of self-renewal and a broad differentiation plasticity
They are derived from pre-implantation embryos and can be propagated as a homogeneous, uncommitted cell population for an almost unlimited period of time
Even after extensive genetic manipulation, mouse ES cells are able to reintegrate fully into viable embryos when injected into a host blastocyst
After these pre-implantation embryos are implanted into a surrogate mother, they develop into mosaic offspring known as chimeras. The tissues of chimeric mice are comprised of a mixture of cells that originated from both the host embryo and the ES cells.
Knockout MiceA knockout mouse is a genetically
engineered mouse in which one or more genes have been turned off through a gene knockout
Important animal models for studying the role of genes which have been sequenced, but have unknown functions
By causing a specific gene to be inactive in the mouse, and observing any differences from normal behaviour or condition, researchers can infer its probable function.
Transgenics This technique permits the introduction of
foreign genes or altered forms of an endogenous gene into an organism
Mostly, this technique does not result in replacement of the endogenous gene, but rather the integration of additional copies of it
The introduced gene is called transgene and the organism carrying it is referred to as transgenic
Transferring DNA into eukaryotic cellsProduction of both knockout and transgenic
animals requires the transfer of DNA into eukaryotic cells.
Calcium precipitationLiposome deliveryMicroinjectionElectroporation
Calcium phosphate precipitates of DNA form when DNA is mixed with calcium chloride. When these DNA precipitates are added to animal cells growing in culture, the precipitated DNA can be taken up by the cells, again transferred to the nucleus and expressed
Liposomes are artificial membranes that can be formed in a test tube. DNA can be mixed with the liposome preparation under the appropriate conditions. This results in the encapsulation of DNA into synthetic lipid membranes. When this membrane fuses with the cell plasmamembrane, DNA is released into the cell and somehow ends up in the nucleus.
DNA microinjectionDNA can also be
injected directly into the nuclei of both cultured cells and developing embryo
Electroporation
Cells are subjected to a brief electric shock of several thousand volts and become transiently permeable to DNA. Presumably the shock briefly opens holes in the cell membrane allowing the DNA to enter the cells before the holes reseal
DNA incorporation in the cellOnce the foreign DNA is inside the host cell,
enzymes that function normally in DNA repair and recombination join the fragments of foreign DNA into the host cells chromosome
The new fragment can either replace an endogenous gene- homologous recombination or it can remain as an independent extrachromosomal DNA molecule referred to as an episome
Identification of transgenic cellsSince only a relatively small fraction of cells
take up DNA, a selective technique must be available to identify the transgenic cells
In most cases the exogenous DNA includes two additional genes
The small fraction of cells in which homologous recombination takes place can be identified by a combination of positive and negative selection
Positive and Negative selectionPositive Selection- One of the additional genes (neoR)
confers neomycin resistance; it permits positive selection of cells in which either homologous (specific) or non-homologous (random) recombination has occurred
Negative selection- The second gene, thymidine kinase gene from Herpes Simplex Virus (tkHSV) confers sensitivity to gancyclovir(a cytotoxic nucleotide analog). This gene permits negative selection of ES cells in which non-homologous recombination has occurred
Only ES cells that undergo homologous recombination (i.e. gene-targeted specific insertion of the DNA construct) can survive this selection scheme
Recombinats with random insertion Nonrecombinat cell
Recombinats with gene-targeted insertion
Treat with neomycin(positive selection)
Treat with gancyclovir(negative selection)
Knockout MiceGene knockout is a technique for selectively
inactivating a gene by replacing it with a mutant allele in an otherwise normal organism (mice)
Knockout mice are a useful model system for studying certain human genetic diseases.
Making knockout miceMutant alleles are introduced by
homologous recombination into Embryonic Stem cells
ES cells containing the knockout mutation are introduced into early mouse embryos. The resultant mice will be chimeras containing tissues derived from both the transplanted ES cells and host cells. These cells can contribute to both germ cell and somatic cell population
Chimeric mice are mated to assess whether the mutation is incorporated into the germline
Chimeric mice each heterozygous for the knockout mutation are mated to produce homozygous knockout mice
Knockout Mice to study genetic diseasesKnockout mice make good model systems for
investigating the nature of genetic diseases and the efficacy of different types of treatment and for developing effective gene therapies to cure these often devastating diseases
For instance, the knockout mice for CFTR gene show symptoms similar to those of humans with cystic fibrosis
Transgenic animalsTransgenic animals carry cloned genes that have
integrated randomly into the host genomeTransgenic technology has numerous
experimental application and potential therapeutic value
The frequency of random integration of exogenous DNA into mouse genome at non-homologous sites is very high, therefore, the production of transgenic mice is a highly efficient and straightforward process
•Foreign DNA containing a gene of interest is injected into one of the two pronuclei (the male and female nuclei contributed by the parent) of a fertilized mouse egg before they fuse•The injected DNA has a good likelihood of being randomly integrated into the chromosome of the diploid zygote
Injected eggs are then transferred to foster mothers in which normal cell growth and differentiation occurs
About 10-30% of progeny will contain the foreign DNA in equal amounts in all tissues, including the germ cells
Immediate breeding and backcrossing of these mice can produce pure transgenic strains homozygous for the transgene
Transgenics and gene therapyOnce a gene mutation is identified to be the cause of
a disease, the next step is to cure the disease by introducing normal genes( transgene) into affected individual
In experimental animals, some genetic disorders have been cured by gene therapy, but in humans, numerous technical issues need to be resolved before it can be widely useda) how to reliably and safely introduce various genes into human cellsb) tissue/ cell specific introduction of genesc) large size of genesd)how to address the ethical issues