Saturday, June 22, 2013

Transgenic Animals and DNA Micro-array

Transgenic animals:
The strategy here is injection of genes in fertilized mouse ovum, these genes will be incorporated into the genome and found in both somatic and germ cells. By this strategy many transgenic animals have been established and are studied for tissue specific effect on gene expression and effects of overproduction of gene products. The transgenic approach has been used to correct a genetic deficiency in mice. Fertilized ova obtained from mice with genetic hypogonadism were injected with DNA containing the coding sequence for the gonadotropin- releasing hormone (GnRH) precursor protein. This gene was expressed and regulated normally in the hypothalamus of certain number of mice, and these animals were in all respects normal.

Their offspring also showed no evidence  of GnRH deficiency. This is therefore evidence of somatic cells expression of the transgene and of its maintenance in germ cells.

There are two broad strategies for transgenesis. The first is direct transgenesis in mammals whereby recombinant DNA is injected directly into the male pronucleus of a recently fertilized egg. This is then raised in a foster mother animal resulting in an offspring that is all transgenic. Selective transgenesis is where the recombinant DNA is transferred into embryo stem (ES) cells. The cells are then cultured in the laboratory and those expressing the desired protein selected and incorporated into the inner cell mass of an early embryo. The resulting transgenic animal is raised in a foster mother but in this case the transgenic animal is a mosaic or chimeric since only a small proportion of the cells will be expressing the protein.

By this method, transgenic goats and cows can now be designed that produce human proteins, like blood clotting factors, in their milk.

Targeted gene disruption or knockout:
Knockout mice are made by creating mutation that totally disrupts the function of a gene. This mutant gene is then used to replace one of two genes in embryonic stem cells creating heterozygous transgenic animal. The mating of two such animals will, by Mendelian genetics, results in a homozygous mutation in 25% of offspring. This can be done by using particular promoter enhancer combinations driving expression of DNA recombinase or miRNAs both of which inactivate gene expression. Such animals can serve as human disease model. E.g. transgenic mice carrying mutant copies of dystrophin gene serve as animal model for study of muscular dystrophy.

Analysis of Gene Expression:

Determination of mRNA level and Protein profiling and Protein-DNA interaction mapping:

Messenger RNA levels are usually determined by the hybridization of labeled probes to either mRNA itself or to cDNA produced from mRNA.

Northern Blot: 
This is similar to Southern Blot  except that the original sample contains a mixture of mRNA molecules that are separated by electrophoresis, then transferred to a membrane and hybridized to radioactive probes. Visualized by autoradiography gives tha amount and size of particular mRNA.

DNA Microarray technology or called DNA chips: 
Here thousands of specific known DNA sequences (synthetic oligonucleotides) are immobilized in glass microscope style slide in few square centimeters. Here target DNA are synthesized chemically or amplified by PCR and UV light cross links DNA to the slide. These are helpful in genotyping and has facilitated to derive entire transcriptome information (entire collection of cellular mRNA) in a few days. A wild type sequence in micro array can be probed with mutant DNA, here hybridization will be reduced and so the signal at relevant spot providing the information on location and type of mutation.

For genotyping analysis, cDNA is synthesized from the population of mRNA from particular cell and labelled with fluorescent tag. This is exposed to gene chip, which is a glass slide or membrane containing thousands of tiny spots of DNA each corresponding to different gene. The amount of fluorescent bound to each spot is a measure of the amount of that particular mRNA in the sample. A microarray can answer which genes are expressed at a given stage of development.

Green fluorescence represents mRNAs more abundant at the single cell stage; red fluorescent represents sequences more abundant later in development. The mRNAs that are equally abundant at both stages of development fluoresce yellow. The spots fluorescence pattern give the snapshot of all the genes being expressed in the cells at the moment they were harvested.

One current application of microarray technology is the generation of a catalogue of SNPs across the human genome. Estimates indicate that there are approximately 10 million SNPs and importantly 200 000 coding or cSNPs that lie within genes and may point to the development of certain diseases.
Next generation sequencing (NGS): 
mRNAs are converted to cDNAs using reverse transcription, and these cDNAs are directly sequenced. This will also help in deriving entire transcriptome information.

Transcriptome information allows predicting the properties and function of proteins expressed in a particular cell, tissue or organ in normal and disease states based upon the mRNAs present in those cells. This is called transcript profiling.

Based on this techniques chromatin immunoprecipitation (ChIP) is developed to map the location, or protein-DNA interaction. Here, chromatin isolated, sheared, and specific protein DNA complexes purified using antibodies that can recognize the protein. DNA bound to this protein is recovered and analyzed using PCR and either gel electrophoresis, direct sequencing (ChIP-SEQ) or microarray analysis (ChIP-chip). Both methods help to identify entire genome- wide locations of a single protein throughout all the chromosomes.

Fig: Chromatin immunoprecipitation technique (ChIP). This method  allows for precise localization of particular protein or modified protein on a particular sequence element in living cells.
(Source: Lehninger's Textbook of Biochemistry and Lippincott's Illustrated Biochemistry)
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