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Detection of Transgenic Animals
#1
This is post no. 1 under the main topic.
Transgenic Animals Detection:

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.

PCR technique:
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.
 
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#2
This is post no. 2 under the main topic.
Other than the method has been described:

I found, there are methods which has been used for making transgenic animal and there are special techniques to identify them. Making of genetically engineered animals by pronuclear injection or retroviral delivery systems has been a successful approach for generating animal models to better understand the functionality of genes. In transgenic animals created by embryo microinjection, the site of integration of the transgene within the genome is a random event. Thus, when multiple embryos have been injected or infected with the same DNA, the integration site will be different in each founder animal. When the integration events have occurred at the one-cell stage, they should exhibit germline transmission with the potential to be inherited by the founder's offspring. If the integration occurs at a later stage, the resulting mosaic founder may or may not exhibit germline transmission of the transgene. In the case of pronuclear injection, there is typically one insertion site, although multiple transgene copies are often found in a tandem array at that integration site.

Lentivirus transgenesis is becoming an increasingly attractive alternative to pronuclear injection because it is more efficient in terms of successful transgene incorporation into the host genome, less invasive to the embryo, and technically less demanding to perform. Lentiviral delivery systems have been used successfully to generate transgenic mice, rats, pigs, and cattle. The disadvantage of lentivirus is that there are often multiple integration events with random transgene insertions on several chromosomes. Independent of the method of transgene delivery, the insertion site can have profound effects on transgene expression. This can lead to phenotypic effects in the transgenic animal that are not due to the transgene per se, but are a consequence of the integration site, a phenomenon referred to as position effect. It is critical to correlate phenotype with genotype, particularly in animals created via lentivirus transgenesis, since not all copies of the transgene may be contributing equally to the phenotype.

Determining transgene integration sites is challenging. A number of PCR-based methods, often referred to as chromosome walking techniques, have been developed to isolate DNA fragments adjacent to known sequences, including inverse PCR, ligation-mediated PCR (LMPCR), randomly primed PCR (RP-PCR), and T-linker PCR. The method I have mentioned gives several elements of these techniques in a unique way that allows the capture of DNA fragments containing the chromosomal region flanking the transgene. These methods enables quick and inexpensive determination of multiple independent transgene integration sites in founder animals and their offspring.
 
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