Archive for the ‘Mouse Tech’ Category


Down Syndrome Mouse

January 18, 2007

Looks like the Down Syndrome Mouse is really starting to pay off

Two papers in the July 6, 2006, Neuron, published by Cell Press, report evidence that surprisingly simple genetic abnormalities in the machinery of critical neuronal growth-regulating molecules can kill neurons in Down’s syndrome, Alzheimer’s disease, and other neurodegenerative disorders. The researchers said their basic findings could aid progress toward treatment for the cognitive deficits in these disorders.

n humans, Down’s syndrome is caused by a trisomy–an abnormal three copies of chromosome 21. Such trisomy causes an increased “dosage” of genes on that chromosome, and a central mystery of Down’s syndrome is how such an overdose of particular genes leads to such abnormalities as mental retardation.

In their papers, Salehi and colleagues and Tessarollo and colleagues studied mice genetically engineered to mimic the trisomy seen in human Down’s syndrome. Their aim was to discover the machinery by which this trisomy ultimately causes the death of neurons that are important for cognitive function.

Salehi et al. find that an increase in the expression of only one gene, for amyloid precursor protein (APP), disrupts transport of the neurotrophin “nerve growth factor” (NGF). APP is also a central molecule in the pathology of Alzheimer’s disease.

The Dorsey et al. paper describes how restoring the normal cellular levels of a Trk receptor for the neurotrophin “brain-derived neurotrophin factor” (BDNF) rescues neuronal death in another mouse model of Down’s syndrome.

Salehi et al. found that the NGF transport disruption leads to the degeneration of “basal forebrain cholinergic neurons” (BFCNs) important for cognitive function. This deterioration of BFCNs is similar to that seen in Alzheimer’s disease and is caused by abnormal APP function. Since in people with Down’s syndrome, the APP gene resides on the trisomic chromosome, Salehi and colleagues reasoned that an overdose of APP might also play a role in neuronal degeneration in Down’s syndrome and thereby contribute to cognitive deficits in both Down’s syndrome and Alzheimer’s disease.



January 14, 2007

Whereas transgenesis involves ‘positive’ or ‘additive’ gene transfer, Gene-targeting procedures are used to disable or ‘knock out’ a candidate gene (23). A construct is designed that has two regions of homology to the target gene, flanking a selectable marker. These markers are bacterial genes that offer resistance to neomycin, hygromycin, or puromycin, which will disrupt the segments of DNA that are either essential for transcription and/or translation. Recombination between the flanking sequences of homology in the targeting construct and the genomic locus replaces the host gene sequence with the selectable marker. [Again the mouse is used as an example animal] The cells that have the selectable marker integrated into their genome are chosen by means of molecular screening techniques prior to being microinjected into blastocysts and implanted into pseudopregnant mice. The heterozygous mice that are produced are mated together to generate homozygous knockout mice, which lack a functional copy of the candidate gene. Gene-targeting is sometimes used to generate mice with a mutation in, rather than inactivation of, the candidate gene. In these cases, a construct identical to the endogenous DNA (other than the desired mutation) is made.

Compared to transgenesis, gene-targeting is an elegant system because it can control both the number of copies as well as the point of insertion. More accurate phenotype modeling is achieved via the absence of insertional mutagenic effects, the complete elimination of the endogenous candidate gene, and its expression in specific cells and tissues. Furthermore, in cases where gene-targeting is used for expressing mutated genes, the mutated gene will be expressed under the same transcriptional patterns as the gene of interest. The experimental advantages of gene-targeting are offset to some degree by its relative labor-intensiveness; transgenesis can yield a stable mouse-line in a matter of months, whereas a gene-targeting line can take years to come to fruition.

Like transgenesis, significant refinement of techniques are necessary before gene-targeting procedures can be applied to therapeutic use in humans.



January 13, 2007

Transgenesis is a technology used to introduce a gene of interest into an animal model for study. [This article will use the mouse as an example animal] Cloned genetic material called a construct is created, for insertion into the mouse embryo. Expression is restricted to a specific tissue by fusing the candidate gene to a cardiac-specific promoter in the transgenic construct. To produce a stable genetic modification, the construct is injected into the pronucleus of a one-cell embryo. The embryo is then implanted into a pseudopregnant female mouse. Mouse pups are genotyped and the transgenic mice, the founders, are mated to produce the stable line (6). Transgenesis is typically used to induce overexpression of transcriptional levels of the candidate gene. However, neither the copy number nor the point of insertion can be controlled, and both the transgene and the native gene are expressed. Consequently, it is necessary for the transgene expression to be dominant over its homologue in order to produce a phenotype. Furthermore, DNA insertion can result in mutagenic effects in the flanking DNA, complicating any resultant phenotype. Hence, several independent founders are generated to check the levels and sites of transgene expression and compare the phenotypes. Transgenesis is typically used to investigate endogenous protein function and signal transduction pathways.

If transgenesis sounds a lot like gene therapy – thats because it is. The process I described is very similar to what could one day be used for germline gene therapy. Because of the expertise gained from generating transgenic mice, germline gene therapy may be more easily achieved than somatic gene therapy. However, the technology is still very crude at this point, and needs to be refined before therapeutic use in humans is possible.


Dr. Frankenstein’s Mouse

January 4, 2007

This is a little out of date, but Dr. Irving Weissman plans to create the first chimera in the United States. He wants to genetically engineer a mouse with human neurons. The resulting transgenic mouse would be useful for testing drugs for schizophrenia, ALS, parkinson’s, alzheimers and other neurological diseases, as each neuron would be completely human. As of now, the plan is to kill the mice if their brain structure or behavior deviates from normal mouse behavior.

I wonder if this will become a big story in the general media.


Schizophrenia Mouse

December 29, 2006

Scientists at John Hopkins have announced the creation of a new researchers admitted that “Rodent models of schizophrenia have significant limitations. The neuronal circuits affected in people are more complex than the analogous circuits in rodents. In particular, the relative size of the prefrontal cortex that is involved in the cognitive deficits is much smaller in rodents than in primates. Some of the cognitive symptoms such as hallucinations or delusions are impossible to address.”

Personally, I can’t get too excited because (as you may already know) I am a big believer in the developmental nature of schizophrenia. But, as always, I wish them luck as I am happy to be proven wrong.


Of Mice and Men – Transgenic Mouse Models

July 15, 2006


Most serious illnesses are complex diseases. Complex diseases are caused by a variety of interacting genetic and environmental elements. Because of their entangled genesis, complex diseases are difficult to understand and treat. For example, diet, exercise, other lifestyle changes, and hereditary factors can all contribute to the development of heart disease.

The recombinant DNA revolution in the 1970s provided the technology for biomedical researchers to investigate the genetic and molecular basis for complex human diseases such as heart disease. Molecular genetic techniques enable scientists to create animal models that mimic various aspects of a disease. Studying the effect of highly specific genetically engineered changes offers insight into the genetic and molecular mechanisms that underlie the onset of disease.

Transgenic Mice in Cardiovascular Disease Research

Recently, the mouse has emerged as the most prevalent experimental model for cardiovascular research. Formerly, larger mammals such as pigs and rabbits had been the preferred experimental animals. Many researchers perceived the differences in cardiac size and morphology to be so large as to preclude the use of mice as a relevant experimental model for human cardiovascular disease. As the comparative ease in applying recombinant DNA technology in mice (relative to other species) became apparent, the popularity of murine experimental models grew. The advantages of short gestation periods, cost-effective maintenance, and a well-characterized genome make the mouse the ideal experimental model. Animal models are useful not only to elucidate the molecular mechanisms that underlie heart disease, but also to test experimental therapies. Recently, the miniaturization of technologies for measuring cardiac endpoints has vastly increased the variety of cardiovascular traits that can be modeled. In 1992, a mouse deficient in apolipoprotein E, the apoE -/- mouse, was generated as the first mouse-line with a stable genetic background that developed spontaneous arterial lesions and coronary artery occlusion (2,3). Since that time, the size and scope of cardiovascular research on genetically engineered mouse models has grown prodigiously.

Transgenic Mice in Psychiatric Research

Psychiatry remains the branch of medicine that has had the least success with developing animal models. Traditionally, rats have been standard test animal for psychiatric research, but recently the popularity of transgenic mice in other areas of medicine has shifted animal research towards mouse models. Most of the focus has been towards developing robust behavioural models of anxiety-related behaviors. Most models rely on evaluating behavioral reaction to stressful situations. Because of the overwhelming, and irreducible complexity of the causes of psychiatric illnessses, researchers have faced difficulty creating translational models for depression or any other mental illness. Consequently the focus has shifted towards generating ‘endophenotypes’ of disease. This means that researchers are shifting their focus towards recreating specific features of disease, with specific genetic or neurological causes.

Because of the wide gulf between human and mouse ‘lifestyles’, I am unconvinced about the degree of usefulness in this approach to psychiatric research – especially because the neurobiologial and genetic bases for psychiatric diseases are still so poorly understood. Future progress in the use of mouse models depends upon improvements in technology for behavioral assessments. Only with detailed, sophisticated and (most importantly) standardized assays for evaluating behavior in mice will the use of mouse models in psychiatric research fully mature.


Psychiatric and cardiovascular disease research represent the two ends of the spectrum with regards to successful incorporation of transgenic mouse models. There remain, however, some concerns that are common to all fields of mouse research. For example, recent evidence suggests that researchers should exercise caution when drawing conclusions from mouse research as the genetic background of specific strains will affect the severity of the phenotype that is expressed (epigenetics anyone?).

Another problem is that position-dependent non-specific effects can be caused when transgenes are insterted randomly into the genome and interrupt existing genes – resulting in an insertion mutation.

Also, transgenic expression of presumably <ectopic proteins> can also compromise results. For example, it has been shown that transgenic expression of <GFP>, normally considered non-reactive, can have diverse phenotypic effects (29). Perhaps even more worrying are the consequences of gene redundancy and compensatory responses (30). Evidence indicates that the molecular events under investigation are part of immensely complex biological processes governed by multiple genetic and molecular influences. Crudely knocking out or overexpressing a protein may activate normally dormant compensatory mechanisms. In such a scenario, the phenotype expressed would not be indicative of a loss of actual function. This sort of difficulty will be eliminated with the emergence of more refined genetic technologies that will allow for more cell type-specific and time-governed genetic manipulation.

Future Directions

There are several collaborative projects underway to improve the resources available to researchers using genetically manipulated models. Two of the most ambitious are the Mouse Phenome Project undertaken by Jackson Laboratories (Bar Harbor, Maine; and a Complex Trait initiative begun by Complex Trait Organization (Memphis, Tennessee; The Mouse Phenome Project is an attempt to create a public database of detailed phenotypic data on the most common inbred strains. They also hope to generate new lines that closely represent human disease through directed breeding based on the natural variation among extant mouse strains and chemically induced, whole-genome mutagenized mice. The creation of the database and the generation of new models will allow researchers to make informed decisions about what background to use for genetic manipulation when making new models. The information will also help scientists sort out strain-dependent effects when interpreting data. Geneticists at the Complex Trait Organization want to locate genes involved in complex traits by creating 1000 new lines of “recombinant inbred” mice. Each line would be a combination of eight existing strains; the scientists would outcross the mice for four generations and then breed brother-sister pairs for 20 generations to create inbred lines. Researchers hope the new lines will be able to reveal the genes that interact to produce complex, non-Mendelian traits and represent the variation in natural mouse populations.

The emergence of functional genomics presents a great new opportunity for research involving genetically manipulated mouse models. New technologies such as gene microarrays let researchers look at the activation of thousands of genes at any given time. This is especially advantageous in the study of complex polygenetic diseases such as cardiovascular disease. As this and other functional genomic technologies mature, researchers will have powerful new tools to examine complex genetic interactions.

Down Syndrome Mouse