In recombinant DNA technology after the gene of interest has been isolated and transferred efficiently, the next major step is to ensure its proper and maximum expression in order to obtain proposed results. Thus in a group of transgenic animals it is important that the animal with proper transgenic integration is identified.
Identification of transgenic animals:
The identification of transgenic animals is done using different mechanism.
In animals, where the transgene produces a distinctive phenotypic effect, the transgenic successful animals can be noted easily. But the results of all the gene transfer does not result in such distinct effects therefore other techniques have to be employed. The dot blot technique and PCR technique are to name a few.
Dots blot technique:
This technique helps in detecting several samples in one experiment. The sample of DNA collected is fixed onto a support like nitro cellulose filter. This then undergoes denaturation so that the double helix separates. Such membrane containing denatured sample when treated with radioactively labelled probe of corresponding transgene, the sample DNA incorporated with transgene binds with the probe. Upon removal of free probes by washing and analysed by autoradiography, it reveals presence of transgene which can be detected by fluorescence produced by radioactive probes. The strength of radioactivity exhibited shows the strength of transgene integration.
This is the most important and wide speed used technology to identify the transgenic successful animals. The primers corresponding to the integrated transgene is used to amplify the test DNA sequences isolated from the transgenic animals. This results in the amplification of the transgene. The amplified DNA when blotted and hybridized, the presence of transgene is confirmed. But the techniques adopted for identification of transgenic animals does not reveal the actual level and site of integration. For achieving these further steps has to be adopted like:
Analysis of transgene integration:
In order to analyse the transgene integrated, the isolated gene samples which are detected with transgene integration, undergo restriction digestion with known restriction endonucleases. The fragments are separated by agarose gel electrophoresis and these fragments are analysed by southern blotting procedure. The fragments on the gel are transferred to a nitrocellulose filter membrane and denatured and probed with radioactive probe to reveal the actual site of transgenic integration. The integration of transgene can be confirmed by choosing the appropriate restriction enzyme. The presence of single or multiple integration of transgene is indicated by southern blotting.
Analysis of mRNA production:
The mRNA produced from a transgene can be detected as it is different from the native mRNA of the organisms. These can be purified and hybridized with a radioactive probe to detect mRNA production.
Analysis of protein expression:
The final aim of transgenic integration is to produce proteins coded by the transgene and its efficient expression. Therefore the detection of transgene integration is possible by protein expression of the transgene. The analysis of transgenic proteins can be done by two methods: by identifying specific antibodies produced by transgenic proteins and by studying the enzymatic properties of the concerned proteins. In the case of antibody identification, various immunologic assays can be used. ELISA test is a classic example of the same. In this case, the antibody specific to the antigenic protein is added and allowed to react. Following the first reaction, this is further reacted with an antibody specific to the former antibody results in formation of a complex. This when treated with the substrate of corresponding enzyme, it produces colour proportional to the strength of the antibody indicating the amount of corresponding transgenic protein expressed. The other assays included are immunoblotting, radioimmunoprecipitation, immunohistochemical staining etc.
Enzyme activities can be detected by using transgenic genes which produces different enzymatic activities or different pathway which is not shown by host enzymes. Various procedures can be employed to detect this. The main procedure followed is by transfer of scorable marker genes. Scorable marker genes are those which produce a definite phenotype. Main examples for these are chloramphenicol acetyltransferase (CAT) genes used mainly in fish or mammal cells, betagalactocidase and luciferase gene used in fish.
All this analysis has to be repeated in two or more different potentially transgenic animals so as to determine the level of transgenic integration as, in different organisms it tend to differ. This occurs because the site of integration may differ among animals. The animals has to be studied to confirm which site integration yield maximum expression and further research must be forwarded to achieve the same.