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The below mentioned article provides notes on gene targeting procedures in biological process.
A mutational approach has proved to be invaluable to investigators examining the roles of gene products in complex biological processes within prokaryotic and cultured eukaryotic cells. Recently it has become possible to apply this approach to a mammalian system. Gene targeting procedures enable the precise (site-specific) introduction of a mutation to one of the estimated 100,000 murine genes.
Typically, mutations have been designed to eliminate gene function, resulting in the generation of “knockout” or “null mutant” mice. The introduction of mutations that produce more subtle alterations in gene function has also been achieved.
Two major developments have made gene targeting experiments feasible:
(a) The generation of totipotent embryonic stem (ES) cells, and
(b) The elucidation of techniques to achieve homologous recombination in mammalian cells.
ES cells are derived from 3.5-day-old mouse embryos, at the blastocyst stage of development. Blastocysts are cultured individually under conditions that permit the proliferation of the inner-cell mass cells, which are those cells that would normally become the foetus. These cells are then disaggregated, and individual ES cells clones are grown.
Under optimal conditions, ES cells retain the ability to contribute to all or the tissues of the developing foetus. The derivation of ES cells was pioneered using embryos derived from the 129/Sv strain of mice, a strain that has been most commonly used in studies of early embryonic development. Although this mouse strain is not ideal for the study of behaviour, most ES cell lines in current use are 129/Sv-derived.
Homologous recombination is the process by which a mutation is targeted to a precise location in the genome. A targeting construct is generated that typically consists of a target gene sequence into which a loss-of-function mutation has been engineered.
Most targeting constructs are designed to achieve homologous recombination events in which recombination at the target locus results in replacement of native target sequences with construct sequences.
In mammalian cells, fragments of DNA preferentially integrate into the genome at random locations, at rates that greatly exceed homologous recombination. Therefore, targeting constructs are designed for use in selection strategies that enrich for ES clones in which homologous recombination has occurred.
In the commonly used positive-negative selection strategy, a portion of a protein-coding exon is replaced by sequences that confer resistance to the drug neomycin.
This mutation serves two purposes:
(a) To inactivate the gene product, and
(b) To provide a marker that enables the selection of cells that have integrated the construct.
This exogenous DNA fragment is flanked by regions of DNA that are homologous to the native gene. Adjacent to one of these homologous regions is a gene encoding thymidine kinase. Treatment with the drug ganciclovir will kill cells that express this gene.
The targeting construct is typically introduced into ES cells by electroporation. In this step, cells are subjected to an electric current that facilitates the internalization of the DNA construct. Those cells that failed to incorporate the targeting construct are killed by the addition of neomycin to the culture medium (positive selection).
The majority of the remaining cells have incorporated the entire DNA construct (including the thymidine kinase gene) at random sites throughout the genome. By contrast, during homologous recombination, non-homologous regions of the construct that are not flanked by homologous sequences are excluded from the integration event. Therefore, homologous recombinant clones will not contain the thymidine kinase gene.
Thus, the addition of a second drug, ganciclovir, will selectively kill cells that have randomly incorporated the construct (negative selection), thereby enriching for targeted clones. ES cell clones that survive this double drug selection are then screened for homologous recombination by PCR or Southern blot analysis.
The homologous recombinant clones, which are heterozygous for the introduced mutation, are then used to generate chimeric mice. Following the isolation of homologous recombinant ES cell clones, these cells are microinjected into the fluid-filled blastocoele cavity of 3.5-day-old embryos at the blastocyst stage.
The injected embryos are then surgically transferred into the uterus of pseudopregnant females. These animals will then give birth to chimeric mice, which are derived partly from the injected ES cells and partly from the host embryo.
For example, ES cells derived from a brown strain of mice are often injected into embryos derived from blackC57BL/6 mice, resulting in chimeras with coats containing black and brown patches.
The extent to which the ES cells have colonized the animal may be roughly approximated by the extent of the brown contribution to the coat. It is most important that ES cell derivatives colonize the germ cells of the chimera, so that the targeted mutation can be propagated to subsequent generations.
If chimeras are mated with C57BL/6 mice, then the germ line transmission of ES cell-derived genetic material is indicated by the generation of brown offspring. Half of these brown mice will be heterozygous for the targeted mutation. These heterozygous mice are then bred to produce homozygous mutant mice that completely lack the normal gene product.
Uses of Gene Targeted Mice:
Studies of null mutant mice provide novel insights into the functional roles of neural genes and, in some cases, animal models relevant to human neuropsychiatry disorders. An illustrative example is a recent study of mice lacking the hypothalamic neuropeptide orexin.
Observations of homozygous mutant mice revealed an unanticipated phenotypic abnormality. The mutants displayed frequent episodes of inactivity characterised by the sudden collapse of the head and buckling of the extremities. Subsequent electroencephalogram (EEG) analysis revealed these episodes to be similar to narcoleptic attacks observed in humans and in a strain of narcoleptic dogs.
Moreover, a mutation of an orexin receptor gene was found to underlie the canine syndrome. Thus, the null mutant phenotype revealed a novel role for orexin in sleep regulation. In addition, this line of mice represents an important animal model for examining the pathophysiology and treatment of narcolepsy.
Another example illustrates the potential utility of null mutant mice to uncover mechanisms underlying the behavioural effects of psychoactive drugs. The non selective serotonin (5-hydroxytryptamine, 5-HT) receptor agonist m- chlorophenylpiperazine (mCPP) interacts with several subtypes of 5-HT receptors.
Although this compound typically reduces locomotors activity in rodents, it produced a paradoxical hyperlocomotor response in a line of 5-HT2C receptor null mutant mice. This response to mCPP was blocked by pre-treatment with a 5-HT1B receptor antagonist, indicating that the absence of 5-HT2C receptors unmasked a hyperlocomotor effect of mCPP on 5-HT1B receptors in mutant mice.
These results provide a model whereby genetic endowment may contribute to the development of a paradoxical drug response. When a compound alters the function of multiple gene products with opposing influences on behaviour, then mutations or allelic variation of these genes may lead to paradoxical effects.
Although gene targeting techniques are most commonly used to generate animals with null mutations, subtle mutations may also be introduced that alter, but do not eliminate, function. The benefits of such an approach are highlighted by a mutation of a gene encoding the α1 subunit of the γ-aminobutyric acid A (GABAA) receptor.
The mutation produced a single amino acid change, rendering the α1 subunit -containing sub-population of GABAA receptors insensitive to benzodiazepines, without affecting their responsiveness to GABA. The resulting animals exhibited reduced sensitivity to the sedative and amnestic effects of diazepam, but no change in sensitivity to the anxiolytic-like effects of this drug.
These results indicate that benzodiazepine site ligands devoid of activity at α1 subunit-containing GABAA receptors may act as anxiolytics devoid of some of the side effects typically associated with benzodiazepines, a prediction borne out by a recent report of the behavioural effects of such a compound. These insights would not have been obtained using a conventional gene targeting approach, because a null mutation of the α1 subunit gene would profoundly perturb brain GABA signalling.
Interpretation of Targeted Mutant Phenotypes:
In interpreting behavioural phenotypes, attention must be paid to the effects of genetic background. The phenotypic consequences of many targeted mutations may be influenced by modifying genes that differ among various inbred strains. In some cases, phenotypic abnormalities have been lost when mutations were bred to a new genetic back-ground.
It may therefore be useful to examine the persistence of mutant phenotypes in the context of several genetic backgrounds. In one example, three groups independently generated lines of mice with null mutations of the 5-HT1A receptor subtype.
Interestingly, although each group placed this mutation on a different genetic background, all observed enhanced anxiogenic-like behaviours in the mutant lines. Thus, particularly strong evidence is provided for a contribution of the 5-HT1A receptor to the regulation of anxiety.
Another potential problem arises from the common use of ES cells derived from 129/Sv mice. Mice of this strain are susceptible to structural abnormalities of the CNS, such as agenesis of the corpus callosum, and are impaired in several behavioural assays.
This potential problem may be addressed through breeding programs to place targeted mutations on different inbred backgrounds, and by the generation of ES cell lines derived from other inbred strains. It has been recommended that the C57BL/6 and DBA strains be used as standards, due to the extensive body of data relating to the behavioural characterisation of these strains.
In addition to the above strain considerations, the standard application of gene targeting technology has several inherent limitations. The null mutations engineered into knockout mice are typically constitutive, i.e., they are present throughout embryonic and postnatal development.
Therefore, the potential for developmental perturbations is a major caveat to the interpretation of mutant phenotypes in adult animals. It may be difficult to determine whether a mutant phenotype reflects a normal adult role for the targeted gene or an indirect effect of the mutation attributable to perturbed development. Such an effect may lead to an overestimation of the functional significance of the gene product in the adult animal.
Conversely, if significant compensation for the loss of a gene product occurs during development, then the severity of the mutant phenotype may underestimate the functional significance of the gene product. The nature of such compensatory mechanisms and the extent to which they exist may be difficult to determine.
The above considerations also pertain to the analysis of transgenic mice carrying constitutive mutations. Another limitation of the standard gene targeting technology is that the mutations are ubiquitous, present in all of the cells of the animal.
Thus, if a neural gene of interest is also expressed in peripheral tissues, then the absence of the gene product peripherally could lead to a lethal or altered phenotype, independent of its neural role. Moreover, for genes that are widely expressed in the CNS, it may also be difficult to anatomically localise the brain region(s) that underlie the mutant phenotypes.