The year of the rat
Break-through in embryonic stem cell technology offers much excitement in the modelling of human disease
Laboratory animals have long been used in medical research as tools to demonstrate the underlying pathological or genetic processes involved in many human diseases. Indeed, the humble lab rat was already being used for this purpose by the mid 19th century, and by the beginning of the 20th century the first inbred rat strains were being developed by Henry Donaldson at the Wistar Institute in Philadelphia. This colony, bred for research into genetics, neuroscience and cancer , was the beginning of a century-long (and still continuing) relationship between scientist and rodent. The benefit gained from this venture over the past 100 years has proved invaluable to many areas of medicine, from models of cardiovascular disease, to transplantation , depression , and more recently the potential of cell therapy to restore function to the body following spinal cord damage .
History of transgenics
Transgenic technology allows a gene of interest to be introduced into the genome of a laboratory animal, and is an extremely powerful tool to pry apart the molecular underpinnings of disease. Transgenics was developed in mice during the 1980's, initially to silence an existing gene (knockout models). Subsequently the technology was developed to introduce a new gene, or have a gene expressed at higher than normal levels (knockin models), and to have a gene expressed in a specific tissue and / or at a specific time (conditional transgenic models) . In mice, this involves using embryonic stem (ES) cells. These are cells extracted from the inner part of an early embryo , and are defined by three special properties:
i) ability to renew themselves indefinitely, and the potential to develop into any cell in the body (pluripotency)
ii) ability to be incorporated into an organism
iii) ability to develop into gametes to be passed onto offspring .
In mice, the ES cells are isolated and maintained in a cell line, where the DNA can be altered by adding, subtracting or modifying genetic material with relative ease. These cells can then be reintroduced into the embryo, where they develop into many of the body’s cells.If they develop into the gametes, when these animals reproduce they will give birth to heterozygote offspring, which are those that have one copy of the introduced gene. If two heterozygotes mate, they will give birth to some offspring with two copies of the introduced gene (homozygotes).
Transgenic mouse models are proving to be very useful, especially for genetic disorders such as Duchenne muscular dystrophy and cystic fibrosis, and are now used to study a wide range of diseases. Indeed, in an evaluation of the efficacy of animal models for the 100 best-selling drugs, Zambrowics& Sands state that “A retrospective evaluation….indicates that these phenotypes correlate well with known drug efficacy, illuminating a productive path forward for discovering future drug targets”.
Whilst this offers excitement and promise to the scientific community, there are some drawbacks, notably that mice have fewer physiological similarities to humans than rats, so are less appealing as models of human conditions. Behaviorally, rats are similar to humans in their ability to learn and accomplish different experimental tasks. Because of their larger size it is also much easier to perform surgical procedures and monitor physiological states in rats than in mice . Until recently, however, attempts to isolate ES cells in rats had proved futile. In mice, the ES cells were maintained in their pluripotent state by keeping them with calf-serum and so-called 'feeder cells' which release a chemical known as Leukemia Inhibitory Factor (LIF) , but this method failed in rats . In animals such as sheep and pigs the problem was overcome using cloning techniques , but again, this proved difficult to accomplish in the rat. The cells appeared to be very unstable, with any disturbance leading them to activate, so that they could not be implanted in an embryo and brought to term.
This is not to say that transgenic rats have never been used in experimentation, but it has meant employing other techniques, not ideal for the experimental purposes . All of these techniques have fundamental drawbacks.
For example, a common method is to introduce random mutations into cells, either using the chemical ENU , or retrotransposons , which are mobile genetic elements that randomly insert into genes , disrupting their activity. The problem with using either of these techniques is that the resulting mutations are random. The scientist needs to mutate a vast number of animals, and screen their resulting phenotypes, then carry out genetic analysis to check whether a change in behaviour is due to a mutation in the desired gene. This is clearly a very expensive procedure.
A different tactic is to take a gene of interest, and incorporate it into the nucleus of a single-celled embryo by direct injection , or by using a lenti-virus to infect the cell with the gene . This method can only be used to introduce small amounts of genetic material - up to about 10kb in length. which is too small to accommodate most genes. It is subject to “position effects”, in other words, it will be inserted into the genome at a random position, so will affect, and be affected by, the genetic material around it.
These problems can be overcome by using artificial chromosomes (such as bacterial, yeast), since they allow a much greater amount of genetic information to be incorporated, and can include a “buffer” region around the gene of interest to protect it from position effects. Unfortunately this method is far less efficient and many lines of transgenic animals often have to be made before it is successful. These problems have meant that the impact of transgenic rats on biomedical research so far has been very limited.
At the end of 2008, two exciting studies were published in the journal Cell that promised to radically change this situation. Two groups independently developed similar techniques to isolate and maintain pluripotent embryonic stem cells in rats: Professor Austin Smith’s lab at Cambridge, UK
Serum, which is used in the methods for isolating and growing mice ES cells, contains substances that drive rat ES cells, and indeed S cells from some mouse strains, towards commitment so the researchers removed it from the culture medium.
However, removal of serum alone is not sufficient to maintain rat tem 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. Promising research in mice which showed that disrupting this pathway allowed the stem-cells to self-renew was published in 2006 so both groups of scientists used a '3 inhibitory' (3i) strategy, which used:
i) an inhibitor-substance to reduce the activity of FGF4
ii) an inhibitor of the MEK pathway to disrupt the signaling
iii) an inhibitor of another substance called glycogen synthasekinase 3 (GSK3) which is used to inhibit the biosynthetic capacity of the cell
Excitingly, both studies found that this technique allowed embryonic stem cells to be kept in a pluripotent state indefinitely. Whilst one of the studies9 found slight problems (sex bias and mutations), a minor modification made the procedure robust and reliable.
The impact on the scientific community is predicted to be enormous. Now, for the first time, ES cells can be used 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 and powerful as is currently the case in mice, yielding better models of human disease. As the Chinese year of the rat wanes, it seems that the time of the lab rat is waxing rapidly.
- Donaldson, H. H. (1912) The history and zoological position of the albino rat, J. Acad. Nat. Sci. (Philadelphia), 15, 365-69.
- Hartcroft, W.S. et al., (1952)Atheromatous changes in aorta, corotid and coronary arties of choline-deficient rats, Proc. Soc. Exp. Biol. Med., 81(2), 384-93.
- Pinto, M. et al., (1983) The relative roles of MHC and non-MHC antigens in bone marrow transplantation in rats: graft acceptance and antigenic expression on donor red blood cells, Transplantation, 35(6), 607-11.
- Overstreet, D.H., (1993). The Flinders sensitive line rats: a genetic animal model of depression, Neurosci. Biobehav. Rev., 17(1), 51-68.
- Rasiman G. and Li Y,, (2007) Repair of neuronal pathways by olfactory ensheathing cells, Nat. Rev. Neurosci., 8(4), 312-319.
- Chen, J. et al., (2001) Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats, Stroke, 32(4), 1005-11.
- Abbott, A. (2004) The Renaissance rat, Nature, 428(6982), 464-6.*
- Brook, F.A. & Gardner, R.L. (1997) The origin and efficient derivation of embryonic stem cells in the mouse, Proc. Natl. Acad. Sci. USA, 94(11), 5709-12.
- Buher, M. et al., (2008) Capture of authentic embryonic stem cells from rat blastocysts, Cell, 135(7),1287-98.**
- 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.
- 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.
- Cozzi J. et al., (2008) Use of genetically modified rat models for translational medicine, Drug Discovery Today, 13(11/12), 488-94.*
- Smith, A.G. et al., (1988) Inhibition of pluripotent embryonic stem cell differentiation by purified peptides, Nature, 336(6200), 688-90.
- Brenin, D. et al., (1997) Rat embryonic stem cells: a progress report, Transplant. Proc., 29(3), 1761-65.
- McCreath, K.J. et al., (2000) Production of gene-targeted sheet by nuclear transfer from cultured somatic cells, Nature, 405(6790), 1066-69.
- Smits, B.M. et al., (2006) Generation of gene knockouts and mutant models in the rat by ENU-driven target-selective mutagenesis, Pharmacogen. Genomics, 16(3), 159-169.
- Ostertag, E.M. et al., (2007) Mutagenesis in rodents using the L1 retrotransposon, genome Bio., 8(Supp 1), S16.
- Wall, R.J. (2001) Pronuclear microinjection, Cloning & Stem Cells, 3(4), 209-20.
- Lois, C. et al. (2002) Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors, Science, 295(5556), 868-72.
- West, A.G. et al., (2002) Insulators: many functions, many mechanisms, Genes Dev., 16(3)
- Chen, S. et al., (2006) Self-renewal of embryonic stem cells by a small molecule, PNAS, 103(46), 17266-71.**
- Li, P. et al., (2008) Germline competent embryonic stem cells derived from rat blastocysts, Cell, 135(7), 1299-1310.**
- Chen, S. et al., (2006) Self-renewal of embryonic stem cells by a small molecule, PNAS, 103(46), 17266-71.**
** Of special interest
* Of some interest