Archive for the ‘Molecular Biology Toolkit’ 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.


Obstacles to Gene Therapy

January 15, 2007
  • Short-lived nature of gene therapy – Before gene therapy can become a permanent cure for any condition, the therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of gene therapy.
  • Immune response – Anytime a foreign object is introduced into human tissues, the immune system is designed to attack the invader. The risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a potential risk. Furthermore, the immune system’s enhanced response to invaders it has seen before makes it difficult for gene therapy to be repeated in patients.
  • Problems with viral vectors – Viruses, while the carrier of choice in most gene therapy studies, present a variety of potential problems to the patient –toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, there is always the fear that the viral vector, once inside the patient, may recover its ability to cause disease.
  • Multigene disorders – Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy. Unfortunately, some the most commonly occurring disorders, such as heart disease, high blood pressure, Alzheimer’s disease, arthritis, and diabetes, are caused by the combined effects of variations in many genes. Multigene or multifactorial disorders such as these would be especially difficult to treat effectively using gene therapy.


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.


Current Status

January 11, 2007

After more than twenty years of gene therapy, there are still no publicly available gene therapy regimens. After early success in (in vitro) experiments, application to humans has proved to be surprisingly problematic. The clinical trials began in 1990, but have been plagued by technical problems ever since. In fact, serious health complications and deaths in a number of recent clinical trials has raised calls for increased regulatory scrutiny over future clinical trails. For example, in January 2003, the FDA placed a temporary halt on all gene therapy trials using retroviral vectors in blood stem cells after two children in a French gene therapy trial developed a type of Leukemia.


  • 1980 – Richard Mulligan, an M.I.T. researcher, shows that genetically engineered mouse-leukemia retroviruses were effective messengers for carrying human genes into mouse DNA.
  • 1989 – Dr. French Anderson, Eli Gilboa and Dr. Michael Blaese win approval from an National Institutes of Health (NIH) advisory panel for a test that would transfer bacterial genes into immune cells of terminal cancer patients. The trial paves the way for dozens of gene-therapy efforts.
  • 1990 – Dr. French Anderson and Michael Blaese perform the world’s first officially approved gene therapy by manipulating human genes. The patient is a 4 year-old girl named Ashanti DeSilva. She inherited a defective gene from both parents and suffered from ADA (adenosine deaminase) deficiency. The scientists introduce millions of Ashanthi’s own white blood cells into her bloodstream that were extracted from Ashanthi’s blood and genetically engineered to contain a corrected (“therapeutic”) copy of the adenosine deaminase gene. The scientists hope these cells will restore Ashanthi’s immune function by producing a normal version of the defective enzyme. The treatment appears to have been a success.
  • 1999 – The sudden death of Jesse Gelsinger, a patient undergoing experimental treatment for a rare liver disorder at the University of Pennsylvania, raises scores of questions about various aspects of gene therapy. Penn officials say Gelsinger’s immune system had a severe inflammatory reaction that caused multiple organs in his body to fail.
  • 1999– NIH discovers that researchers did not report 6 gene therapy patient deaths. Public backlash intensifies
  • 2000 – Reporters uncover hundreds of unreported cases of “adverse effects” for gene therapy trail.
  • 2001 – First germline gene transfer – 30 children born as a result of ooplasmic transfer. Ooplasmic transfer (also referred to as ooplasmic or cytoplasmic transplantation) is a fertility procedure used by women who cannot conceive because of defects in their ooplasm – their eggs’ cytoplasm. The procedure is performed by inserting healthy ooplasm from donor eggs into the eggs with defective ooplasm. By inserting healthy ooplasm from the donor eggs into the mother’s defective eggs, a small amount of mitochondrial DNA is transferred into the egg. This is considered germline gene transfer because the mitochondrial DNA of these children, and of their offspring, will always be from the donating mother.
  • 2002 – Two children who were cured of “bubble baby syndrome” (X-SCID), were discovered to have developed a leukemia-type disease.
  • 2003 – FDA temporarily halts gene therapy trials using retroviral vectors in blood stem cells. This is the first restriction of government regulation of gene therapy trails since they were allowed in 1989.

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.