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Post details: The Year of the Rat ends on a high note

08/02/09

Permalink 11:30:04 am, by Tom, 987 words, 3442 views   English (UK)
Categories: Information

The Year of the Rat ends on a high note

Transgenic animal technology allows scientists to study the molecular basis of disease, and since the first transgenic mice were produced in the 1980’s it has become a mainstay of biomedical research. Early efforts focused on silencing an existing gene (knockout models), but subsequently methods were developed to introduce a new gene (knockin models), and to express the new gene in a specific tissue and/or at a specific time (conditional transgenic models). In mice transgenic techniques rely on the manipulation of embryonic stem (ES) cells, which have three main properties: i) ability to renew themselves and develop into any cell in the body (pluripotency); ii) ability to be incorporated into an organism; and iii) ability to develop into germ cells to be passed onto offspring. These cells can be modified genetically very easily, and can be reintroduced into the embryo and will develop into many of the body’s cells. Transgenic mouse models are proving to be very useful, especially for genetic disorders such as Duchenne muscular dystrophy and cystic fibrosis. Indeed, in an evaluation of the efficacy of mouse knockout models of genes that are the targets for the 100 best-selling drugs, Zambrowics & Sands (1) note that “A retrospective evaluation….indicates that these phenotypes correlate well with known drug efficacy, illuminating a productive path forward for discovering future drug targets”. It is perhaps not surprising that the Nobel Prize in Physiology or Medicine for 2007 went to Sir Martin Evans, Professor Mario Capecchi and Professor Oliver Smithies for "their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells".

Whilst transgenic mice offer much to the scientific community, scientists have long desired to create transgenic rat models of disease. Behaviorally, rats are much more similar to humans in their ability to learn to accomplish different experimental tasks, and because of their larger size it is much easier to perform surgical procedures and monitor physiological states (2). However, attempts to produce transgenic rats have been severely hampered by the inability to isolate rat ES cells and grow them in vitro. This problem was overcome in some animals such as sheep and pigs by cloning, but again, when applied to the rat, this approach proved exceptionally difficult to perform. This is not to say that transgenic rats have never been used in experimentation, but it has meant that other techniques have had to be employed, all with fundamental drawbacks, meaning they are not ideal for experimental purposes. One of the more popular approaches, for example, takes a gene of interest, and incorporates it into the nucleus of a single-celled embryo by direct injection (3), or by using a lentivirus to infect the cell with the gene (4). This methods suffers from the fact that it is limited to genetic material of about 10kb in length, too small to accommodate most genes, and it is subject to “position effects”, in other words it will affect, and be affected by, the genetic material around it when it is introduced to the genome, because it will be inserted at a random position in the genome. For these reasons the impact of transgenic rats on biomedical research has been very limited.

At the end of 2008, though, two groups independently developed a very similar method to isolate and maintain pluripotent embryonic stem cells in rats: Professor Austin Smith’s lab at Cambridge, UK (5) and Professor Qi-Long Ying’s lab at the University of Southern California (6). Serum, which is used in the protocol for isolating mice ES cells, contains substances that drive rat ES cells towards commitment to a particular cell lineage. Removal of serum alone from the medium is not sufficient to maintain stem cells because the rat ES cells themselves secrete a protein known as fibroblast growth factor 4 (FGF4) that also drives them to commitment, through a cell signaling system known as the MEK/ERK pathway. Earlier research in mice had already shown that inhibiting this pathway maintains self-renewal in stem cells, so both groups of scientists used a 3 inhibitory (3i) strategy, which used i) an inhibitor of FGF4, ii) an inhibitor of the MEK pathway, and iii) and an inhibitor of glycogen synthase kinase 3 (GSK3) - an enzyme that inhibits the biosynthetic capacity of the cells and therefore their ability to proliferate. Excitingly, both studies found that rat ES cells could be kept in a pluripotent state indefinitely, displaying the 3 main characteristics of stem cells, and express certain protein such as Oct4 and nanog that are characteristic markers of stem cells. Whilst one of the studies (5) initially encountered problems, bias in the sex ratio of the offspring and in some cases trisomy on chromosome 9, a minor modification to the procedure made it robust and reliable.

The impact of this work on medical research is predicted to be enormous, as now for the first time ES cells can be used as a method to create transgenic rat models where the modified gene directly replaces the normal gene, overcoming the problems associated with rat transgenics up until now. Using this method, rat transgenics should become as precise, robust and powerful as is currently the case in mice, whilst yielding better models of human disease. The year of the rat may be over, but a new era for rats in research is only just beginning.

Matthew Evans

References

1. Zambrowics, B. P. & Sands, A. T. (2003) Knockouts model the 100 best-selling drugs – will they model the next 100? Nat. Rev. Drug Disc., 2(1), 38-51.

2. Jacob, H.J. & Kwitek, A.E. (2002) Multifactorial genetics: Rat genics: attaching physiology and pharmacology to the genome, Nat. Rev. Gen., 3(1), 33-42.

3. Wall, R.J. (2001) Pronuclear microinjection, Cloning & Stem Cells, 3(4), 209-220.

4. Lois, C. et al. (2002) Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors, Science, 295(5556), 868-872

5. Buher, M. et al., (2008) Capture of authentic embryonic stem cells from rat blastocysts, Cell, 135,1287-1298.

6. Li, P. et al., (2008) Germline competent embryonic stem cells derived from rat blastocysts, Cell, 135(7), 1299-1310. doi:10.1016/j.cell.2008.12.006

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