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In this article we will discuss about:- 1. Subject-Matter of Transgenic Mice 2. Production of Transgenic Mice 3. Uses of Transgenic Mice.
Subject-Matter of Transgenic Mice:
The ability to insert an exogenous (or foreign) gene into the mouse genome by direct injection into the pronuclei of zygotes was achieved just two decades ago. The term transgenic was applied to mice expressing exogenous DNA that had been produced using this technique.
With this method, the gene of interest is inserted into a random locus in the mouse genome, and is expressed “in trans”, i.e., not in its usual genetic locus.
The techniques required for introducing transgenes into the mouse genome have been highly refined, permitting their widespread use. Since the development of this technique, many thousands of lines transgenic mice have been generated, and it has been the most widely utilised technique of genetic manipulation mice.
Production of Transgenic Mice:
Techniques for producing transgenic mice involve the microinjection of DNA constructs into fertilised mouse eggs. DNA constructs used for the generation of transgenic mice typically consist of a gene of interest located 3′ to promoter sequences selected to produce a desired distribution of gene expression.
The maximum length of the DNA sequence that may be successfully incorporated into the mouse genome is not known, and up to 70 kilobase (kb) DNA fragments have been successfully integrated.
The transgene is linearized and purified from prokaryotic vector sequences. For optimal integration efficiency, about 1 to 2 picoliter (pL) of DNA at a concentration of 1 to 2 ng/µL (corresponding to a few hundred molecules of a 5-kb DNA fragment) is microinjected into the male pronucleus of a fertilized mouse egg.
Although labour intensive, direct injection of DNA into the pronucleus results in much higher rates of integration of transgenes than other known methods of transformation. After microinjection, the embryos are surgically transferred into the oviduct of pseudopregnant mice. Pseudopregnant females are generated by mattings with vasectomised males.
The act of copulation initiates the endocrine changes of pregnancy, providing a suitable uterine environment for the survival and implantation of the transferred embryos’. The foster mothers give birth 19 to 21 days after oviduct transfer. For genotyping, DNA is typically isolated from mouse tail biopsies and screened for the presence of the transgene by Southern blotting or polymerase chain reaction (PCR).
Typically, about 20% to 40% of the mice that develop to term possess the transgene. In the majority of cases, integration of the transgene occurs during the one-cell stage, so that the transgene is present in every cell of the transgenic mouse.
Integration usually occurs at a single random chromosomal location, and, for reasons that are not fully understood, there are usually multiple copies of the transgene inserted as head-to-tail concatamers. Mice identified to possess the integrated transgene are referred to as founders. The founders are typically used in a breeding strategy to produce animals that are homozygous for the transgene insertion.
Uses of Transgenic Mice:
Because transgenic mice often possess multiple copies of the transgene, this method can be used to produce animals with increased levels of expression of particular genes, i.e., mice that “overexpress” genes of interest. In addition, it can be used to express altered forms of a gene product in the distribution of the endogenous gene.
One example is a transgenic line bearing a transgene composed of the Ca2+/calmodulindependent protein kinase α subunit (CaMKIIα) promoter driving expression of a mutant form of CaMKIIα that conferred Ca2+-independent activation. These mice exhibited an increased stimulation threshold for the induction of synaptic plasticity in the hippocampus, as well as deficits in spatial memory.
Studies of these animals led to an enhanced understanding of the role of CaMKII_ in synaptic plasticity and spatial memory acquisition. In many cases, it is desirable to express a gene with an anatomic distribution that does not mirror its native expression pattern in the mouse.
Such ectopic expression of a gene may be achieved using a transgenic construct in which the gene of interest is preceded by promoter elements that direct expression in an anatomic distribution characteristic of another gene.
An example of this approach is a transgenic line in which the D1 dopamine receptor promoter was used to drive expression of a cholera toxin subunit (which constitutively activates Gs) in cells that express D1 dopamine receptors.
Studies of these animals revealed that chronic overstimulation (by constitutively activated Gs) of forebrain neurons expressing D1 receptors results in an abnormal behavioural phenotype that was likened to human compulsive behaviours. For most genes, the promoter elements necessary to reproduce the native patterns of expression are not well defined.
A useful approach for identifying important promoter elements for genes of interest involves the generation of transgenic mice in which putative promoter sequences are used to direct expression of reporter genes, whose expression is readily determined in brain tissue. Comparisons may then be made between the pattern of reporter gene expression and that of the gene of interest.
It is also possible use transgenic approaches to reduce the expression of a particular gene product. This may be achieved using “dominant-negative” mutations, mutations that induce loss of function of a gene product when expressed in the heterozygous state.
For example, transgenic constructs may be designed to express antisense RNAs that hybridize to native messenger RNA (mRNA) sequences, thus decreasing production of the gene product of interest.
Alternatively, the function of gene products that aggregate into multimeric complexes may be disrupted by dominant-negative mutations that produce dysfunctional subunits of the complex. The most prevalent approach used to generate loss of function mutations, gene targeting.
Interpretation of Transgenic Mouse Phenotypes:
An important factor that frequently complicates the interpretation of studies with transgenic mice is the difficulty that may be encountered in achieving a desired anatomic distribution of transgene expression. Promoter elements are often quite large, and additional regulatory elements may at times be located great distances from the gene of interest.
In addition, the site of integration often affects the pattern and level of transgene expression, so that founder mice generated with a common targeting construct may display different expression patterns. It may therefore be difficult to accurately duplicate a promoter’s endogenous pattern of gene expression in the setting of a transgenic mouse.
Commonly, expression patterns are assessed in multiple founders, and those with the most appropriate transgene expression would then be selected for a particular experiment. Several additional considerations in the interpretation of phenotypes in transgenic mice warrant mention. For example, the number of copies of the transgene incorporated into the genome varies between founder mice.
In some cases, concatamers can be unstable and susceptible to deletion of one or more copies of the transgene. In addition, the integration of the transgene may occasionally disrupt an endogenous gene. This could lead to the development of a phenotypic abnormality unrelated to the function of the transgene-this is estimated to occur in 5% to 10% of transgenic mice.
This possibility may be assessed by determining whether similar phenotypes are present in animals derived independently from different founders because the likelihood of two founders possessing the same transgene integration site is minimal.