Methods Used in Experimental Animals

Last updated on September 16, 2010

Alternatives to use of animals
    ICCVAM

    Ames test

    Limulus amebocyte assay

    Monoclonal antibody production

Transgenic animal technology 

Homologous recombination

Chemical mutagenesis

Behavior testing

    Activity and behavior testing in rodents

Euthanasia

Alternatives to death as an endpoint

Refinements

    Reducing stress during blood collection
    Gavage in rodents
    Use of radiotelemetry devices

Potential adverse effects of research methods

Alternatives (Replacement)

In 1985, Index Medicus introduced the term "Animal Testing Alternatives" as a subject heading for searches. This addition reflects the importance placed on the use or exploration of alternatives to the use of live animals in research. The estimated increase in cost of maintenance of animals used in research was 500% between 1972 and 1988. One evaluation of how animal use for research has changed revealed the following:{4099}

Alternative testing

The Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM) was originally established in 1993 by the NIH Revitalization Act to promote and expedite the use of new tests that would improve prediction of adverse effects, improve animal welfare, and reduce animal use in research. In 2000 it was signed into law (the ICCVAM Authorization Act of 2000, Public Law #106-545). This law gives permanent standing to the ICCVAM. Its functions are to review petitions for acceptance of alternatives to animal tests, and recommend changes to the appropriate federal regulations. As of 2001, it had recommended the Corrositex kit (a non-animal system for skin corrosion screening) and the murine local lymph node assay.{4603}

Ames test

This test uses bacteria to determine toxicity. It is 80% reliable compared with animal use for the identification of mutagenicity.{4099}

Limulus amebocyte assay

Monoclonal antibody production

"There is evidence that the mouse ascites method of monoclonal antibody production causes discomfort, distress, or pain. Practical in vitro methods exist which can replace the ascites method in many experimental applications without compromising the aims of the study.

"Accordingly, IACUCs are expected to critically evaluate the proposed use of the mouse ascites method. Prior to approval of proposals which include the mouse ascites method, IACUCs must determine that (i) the proposed use is scientifically justified, (ii) methods that avoid or minimize discomfort, distress, and pain (including in vitro methods) have been considered, and (iii) the latter have been found unsuitable...

"The federal mandate to avoid or minimize discomfort, pain, and distress in experimental animals, consistent with sound scientific practices, is, for all practical purposes, synonymous with a requirement to consider alternative methods that reduce, refine, or replace the use of animals..."{3757}

Generation of monoclonal antibodies (MAb) using animals typically involves two phases: (1) creation of a hybridoma and (2) production of antibodies from the hybridoma. The technique was discovered in the 1970s by Köhler and Milstein, earning them the Nobel Prize. A mouse is immunized with an antigen, and then a splenocyte from that mouse is fused with a mouse plasmacytoma cell. The resulting hybridoma is an immortalized cell that produces specific antibody. Although monoclonal antibodies are now produced in large quantities by commercial companies, research needs for monoclonal antibodies fall generally into the category of "a large number, but small quantities (<0.5g)". For clinical use, the humanized MAb Herceptin®, which is directed against a protein in breast cancer cells, is now approved for use.{4021}

To produce the initial hybridoma, mice are immunized with the antigen, and their spleens are collected. B-cell splenocytes are isolated and fused with plasmacytoma cells using polyethylene glycol. Different fused cells are cloned using tissue culture technique and tested for antibody production; selected clones are expanded using in vitro or in vivo techniques to produce the desired MAb. A few laboratories have the ability to skip the use of mice altogether, and create the hybridomas using phage display and in vitro immunization.{4021}

Heterohybridomas are created by fusing splenocytes from a non-mouse species with murine plasmacytoma cells. In vivo MAb production then requires use of an immunocompromised host animal.{4021}

Monoclonal antibody production in vivo

A number of European countries have restricted or prohibited the use of ascites as a means of MAb production in rodents. In the US, OLAW produced a "Dear Colleague" letter advising IACUCs that they have the responsibility to determine whether investigators have considered methods that avoid or minimize pain or distress in animals, and that they use alternative methods if suitable.{4021}

Monoclonal antibody production in vitro

Cells may be cultured using a variety of systems. Stationary culture uses T flasks. Suspension cultures use roller bottles or spinner flasks, providing better gas exchange but lower cell numbers due to shear forces. Perfusion cell culture systems are hollow fiber bioreactors, in which cells are maintained in compartments separated from media by a selective membrane and bathed continuously in media. High-density cell cultures are obtainable. The health of cultures is monitored, particularly in perfusion systems, using pH, nutrient or waste concentrations and cell concentration and viability. Harvest of the antibody involves purification processes such as ammonium sulfate precipitation, affinity or ion-exchange chromatography, and gel filtration.{4021}

Top of page

Transgenic Technology{3929}

Transgenic mutations are dominant, unlike targeted mutations which are usually recessive. Usually, multiple founders are generated and are then maintained using any of a number of breeding or preservation schemes. Homozygotes carrying cancer genes are usually not viable, and are maintained by forced heterozygosity (+/+ x Tg/+) such that only 50% of the offspring are transgenic.{4530}

There were several important advances in reproductive biology without which modern transgenic methods would have been useless. For example, embryo transfer dates back to 1891. By the 1940s scientists were able to sustain fertilized embryos in vitro through several cleavages. These were followed by techniques of mixing mouse embryos to produce chimeras, transfer of inner mass cells and teratocarcinoma cells, and nuclear transfer. Gurdon transferred mRNA and DNA into Xenopus eggs in 1977. Brinster et al did the same thing in mouse ova in 1980, using rabbit globin mRNA. The first published methods of DNA microinjection were produced in 1966 by Lin, but 15 years passed until the first transgenic animals were created. These were mice harboring the growth hormone fusion construct, produced by Palmiter in 1982. Gordon and Ruddle introduced the new term "transgenic" in 1981. In addition to mice, transgenic rats, rabbits, swine, sheep, goats, cattle, poultry and fish have been produced.

There are only three basic transgenic techniques: (1) DNA microinjection, (2) retroviral mediated transfer, and (3) stem cell transfer. Molecular cloning is considered a separate technique. DNA microinjection is by far the most popular technique, acting as the first step in delineating potential directions for further research. Microinjection is useful when the major goal is studying the expression of new genetic information. It can be performed only on single-cell embryos. Retroviral transfer is used to study cell lineage and random insertional mutagenesis, and can be performed anytime from the one-cell stage to mid-gestation. They have received much attention because of their potential use for gene therapy. ES cells are used for site-directed mutagenesis by homologous recombination, which cannot be accomplished by the other two methods. ES cells with the DNA of interest already integrated can be detected prior to their use to generate transgenic mice, because the frequency of integration is relatively high.

DNA microinjection

DNA preparation and purification: Very large fragments (hundreds of kb) can be implanted, using yeast artificial chromosomes (YAC) and P1 vectors. Bacterial artificial chromosomes (BACs) are also available{4529}. Alternatively, overlapping fragments may be used. Important considerations include: (a) linear DNA integrates more efficiently than supercoiled; (b) use of low ionic strength microinjection buffer such as 10mM Tris; (c) DNA concentration of 1-2ng/µl (higher concentrations will integrate better, but egg viability drops); (d) dissimilar ends of the DNA vs. blunt ends; (e) injection of DNA into the male pronucleus is more efficient than into the female pronucleus; and (f) nuclear injection of DNA is much better than cytoplasmic injection. The DNA must be as pure as possible, free of nicks and strand breaks. The best quality reagents, including deionized and RO purified water, must be used. Plasmid DNA is replicated in E. coli hosts, then purified away from any vector sequences on cesium chloride gradients and separated on agarose gel. The final DNA must be free of salts, organic solvents, and agarose. 

Superovulation, egg culture and harvest: Equipment needed to manipulate eggs includes a dissecting stereo zoom microscope with differential interference contrast (DIC, either Nomarski or Smith) optics and 180-400X magnification, micromanipulators, microinjection systems (either air-driven or oil-driven), warming plate, flowmeters to control the atmosphere to 5% O₂, 5% CO₂, 90% N, and glass pipettes produced with a pipette puller and polished with a microforge. Media for egg culture include bicarbonate-buffered media such as BMOC-3, M16, SECM or M2, which are used in the controlled atmosphere, and HEPES-buffered medium to maintain the pH when the eggs cannot be kept in the controlled atmosphere.

Hybrid mice are used if possible, particularly C57BL/6 x SJL F1, because their eggs are of better quality. However, sometimes it is important to have the transgenic mouse on an inbred background, in which case either FVB or C57BL/6 are popular. C57BL/6 have been used for so long that many mutants are already available. Young C57BL/6 females superovulate well and their blastocysts are excellent recipients for ES cells. FVB eggs have large distinctive pronuclei which are easier to inject. Strain 129/Sv are often used to generate ES cells. Outbred ICR or CD-1 mice are often used as recipients for the embryos.

Because it is easier to produce the transgenic on a hybrid strain, an alternative is then to backcross the founder transgenic mouse to inbred strains.

To superovulate mice, PMSG and HCG are given at 5-7.5 U ip to female mice 3-8 weeks of age. Give PMSG at noon on the first day, HCG 24-26 hours later depending on the mouse strain. The females should produce 20-35 eggs. An alternative method is to deliver FSH using Alzet minipumps, then induce ovulation with HCG or LH. This method is more expensive, but may be needed for strains that do not respond well to the PMSG/HCG routine, or in old obese females. After receiving the HCG, females are placed in the male’s cage and checked for copulatory plugs the next day. Eggs should be harvested 8-9 hours after the midpoint of the dark cycle. The mouse is euthanatized and the oviducts removed through a ventral or dorsolateral approach. The eggs are contained within the ampulla of the removed oviducts, which is torn using a small needle to release the egg mass and cumulus cells which surround it. The cumulus cells are digested with a brief exposure to hyaluronidase. Eggs are then picked up with a micropipette, placed in two washes of medium without hyaluronidase, then the best eggs selected and placed in microdrops of medium until ready for microinjection. 

Microinjection: The window of opportunity to inject eggs is usually 3-4 hours, and lasts from the time male pronuclei can be identified until the male and female pronuclei merge just prior to the first cleavage division. The length of the window varies between strains, as just about everything else does, and must be determined with practice. Eggs can be kept for several days in vitro, with blastocysts developing in 4 days and inner cell masses appearing 3 days later. Before performing microinjection the eggs are placed in a drop of injection medium containing cytochalasin B, which stiffens the egg membrane to help prevent cell lysis as the membrane is penetrated by the injection pipette. The holding pipette holds the egg in place with the male pronucleus closest to the injection needle. DNA is constantly running out of the tip of the injection needle, making it necessary to work quickly to penetrate the zona pellucida and pronuclear membrane. The pronucleus is filled with DNA until expansion stops. Eggs should be left on the slide no more than 30 minutes, after which they are transferred through 2 washes of culture medium without cytochalasin B. Routinely, 10-25% of eggs will lyse during this process. Zygotes can be cultured to the 2-cell blastocyst stage, then implanted into the oviduct on day 1 of the recipient’s pseudopregnancy.

Egg transfer: Tribromoethanol should not be used for anesthesia, as it causes peritoneal irritation and fibrinous serositis on a number of abdominal organs. Ketamine/xylazine is better{3539}. A pool of female mice are maintained and observed for proestrus. The day after mating to vasectomized males, the microinjected zygotes are implanted through a dorsolateral approach using a dissecting microscope. 25-30 injected ova are expelled into the ostium of the oviduct and blown gently through to the ampulla. After birth, DNA slot blot, PCR or Southern blots are used to identify pups harboring injected DNA.

If the transgene is inserted into the sex chromosomes, a test mating of a transgenic male to a nontransgenic female will determine which chromosome carries the transgene. If the transgene is on the Y chromosome, only the male offspring will be transgenic. If the transgene is on the X chromosome, only the female offspring will be transgenic.

Top

Homologous Recombination (targeted mutagenesis)

Preplanned genetic alterations can be made in cells by using ES cells, and this is the focus of most of the ES cell work. The DNA combines by homologous recombination with the host cell DNA into a specific site. There is likely no limit to the size of DNA construct that can be inserted. ES cells are obtained from the inner cell mass of mouse blastocysts using a variety of cell culture conditions. They are pluripotent cells. They require complicated feeder cell layers to keep them from further differentiation during the establishment of the cell lines. If this is not done, they will develop into embryoid bodies. If these are injected subcutaneously into syngeneic or immunodeficient mice, teratocarcinomas will develop containing cartilage, secretory epithelium, glandular structures, keratinizing epithelium, melanin, and muscle.

Mouse chimeras are produced by injecting ES cells into the host embryo. Most labs inject the blastocyst stage, but if the technician is good, the morula stage can be injected. Brinster claims to have successfully injected a mouse egg, but got only one offspring from 10,000 eggs. Often, coat colors are used to follow the success of the procedure. ES cells from a pigmented mouse combined with an albino will result in spotty chimeras. When backcrossed to the albino strain, pigmented mice are assumed to carry the ES genes.

Knock-out mice are those that have a loss of function induced by homologous recombination. Knock-in mice have a gain of function. Insertional mutagenesis is the insertion of a transgene such that it interrupts a gene; this may uncover deleterious recessive genes. A new twist, chromosomal engineering, involves making extremely large (centiMorgan) alterations in the genome. The researcher must realize, however, that there will be huge variations in the phenotype of these mice depending on the background strain that was used. {4529} Strains are usually maintained as homozygotes (if they are fertile) or by forced heterozygosity (if they are infertile).{4530}

A very large number of mice are needed to establish an ES cell laboratory. ES cell lines require a census of about 30; pseudopregnant mice are produced using a census of 100 females and 20 vasectomized males to produce the 40 mice/week needed; blastocyst production requires 200 mice/month; embryonic fibroblasts for feeder cells requires 16; and testing the product mice for the inserted gene requires another 100. A total of almost 800 mice are therefore required, plus 100 more per germ line studied. Another author stated that the overall efficiency of production is about 5% of the eggs injected, with most of the losses occurring between embryo transfer (90% survival) and live birth (25%).{4529}

Viral Gene Transfer

Retroviral-mediated gene transfer has the advantage that the inserted gene is integrated stably into the genome, in a single but random location, and the efficiency of gene transfer approaches 100%. Modified Moloney murine leukemia virus (MuLV) is often used in mice. It would also be nice to be able to infect embryos directly with retroviral vectors, but retroviruses are very tissue-specific and single insertions do not occur very often this way.

One way in which retroviral vectors have been used is by the injection of transgenic bone marrow cells into lethally-irradiated recipients. If successful, the new cells can be found in the peripheral blood, Kupffer cells in the liver, microglia, and elsewhere. Research into cell fate and lineage can be performed using this technique. In addition to mice, dogs and NHPs have been successfully used. The dose of radiation differs among mouse strains; 1000 rads are used for C57BL/6, but only 750 rads for BALB/cByJ mice. Southern blots are performed on tissues of the recipients to determine how efficient the transfer of the gene was. 

In human gene therapy, it would be preferable to simply inject the gene vectors without having to remove and re-implant cells; in effect this is a "local transformation." Derivatives of the Moloney MuLV have been investigated for this purpose. Unfortunately, retroviruses only infect actively-dividing cells, which limits the tissues that can be targeted. The lungs would make a dandy place to insert DNA, for example in diseases such as cystic fibrosis and a 1-antitrypsin. Adenoviruses, which infect even non-dividing pulmonary cells, have been used to carry human a 1-antitrypsin into the respiratory tract of the cotton rat, Sigmodon hispidus, where it was expressed for about a week. The same investigator successfully infected cotton rats with the CF gene for a short period. Concerns relate to the potential for adenovirus to recombine with other viruses, and the effects of uncontrolled expression of the gene product. Next, herpesviruses have been used as transgene vectors for human hypoxanthine-guanine phosphoribosyltransferase (HPRT) which may provide a treatment for Lesch-Nyhan syndrome, which cannot be treated by parenteral administration of the protein. Neurons must be targeted for the HPRT to be effective. 

Lentiviruses have now also been used to introduce a small segment of DNA into both mice and rats (reported in Science 295:868-872, 2002). Recombinant lentivirus is injected into the mouse embryo just under the zona, which is fairly easy to do. 80% of the resulting mouse pups were transgenic, with 90% expressing the gene in a tissue-specific manner.{4529}

Conditional Genetics

If the gene of interest is lethal, it is easier to produce affected offspring if the transgene can be maintained in some non-lethal state until it is needed, at which point it can be induced using some relatively easy control scheme. There are at least two such schemes in common use. The Cre/loxP system involves two short segments (loxP) inserted at either end. Depending upon their relative orientation, they may snip out the gene when activated by Cre recombinase (not sure I've got this quite right here!). A knockout mouse line with "floxed" alleles is crossed with a "tool mouse" that is transgenic for Cre to exert the effect in the progeny. A similar system is the Flp/Frt system which is based on the Flp yeast recombinase. One such lab site, including a database of some 7000 entries of cre and floxed mice, is located at http://www.mshri.on.ca/nagy. Transgenes can also be turned on or off using a tetracycline system.{4529}

Binary transgenic systems are those in which the expression of one transgene activates expression of another, with the genes maintained in two separate strains of mice. This is also used for lethal transgenes. There are many activator/responder systems, including an activator consisting of MMTV promoting GAL4 and a responder of UAS promoter driving Int2, a proto-oncogene.{4529}

Top

Somatic Cell Transformation

Although it would be advantageous to create transgenic hepatocytes, these cells are not mitotically very active, are resistant to culture, and delivery to the recipient is problematic. In two rat models, however, some success has been obtained by delivering the transgenic cells on collagen-coated dextran microbeads. Hyperbilirubinemic mutant Wistar Gunn rats have a deficiency of bilirubin-uridine diphosphate glucuronosyltransferase (UDPGT). Analbuminemic Nagase rats synthesize only trace amounts of bilirubin. Both these conditions improved after giving them normal primary rat hepatocytes on microbeads.

RNA and DNA plasmids have been injected directly into muscle cells and appeared to have integrated into the cellular DNA, or at least been able to function for up to 60 days. This has great potential for human therapy. Another treatment for cystic fibrosis involves aerosol delivery of rhDNase I to patients; it breaks down the DNA that forms much of their respiratory detritus.

There are many potential applications of somatic cell transformation with transgenes, either by in situ or ex vivo methods. Myoblasts represent good cells because they can fuse with the recipient myofibers. For example, the mdx mouse has muscular dystrophy. The introduction of normal mouse myoblasts has successfully resulted in dystrophin production. This has also been tried in human Duchenne’s muscular dystrophy patients. Vascular endothelial cells normally secrete several products, and grafts containing transgenic endothelial cells have been placed in dogs, minipigs, and rabbits. Fibroblasts have been made to express several transgene products, including the expression by the Watanabe rabbit of LDL receptors, thus correcting their defect. Long-term induction of immune response to transgenic cells is being worked on; at present cyclosporin A may still have to be given to prevent it. Since the brain is an immunologically privileged site, fibroblasts expressing nerve growth factor have been successfully implanted in rats.

Other methods of creating transgenic mice are discussed in Pinkert’s book. For example, creation of sperm cells with DNA would make microinjection and embryo transfer obsolete. Rabbit sperm have been successfully infected by SV40 DNA and carried into oocytes by either artificial insemination or in vitro fertilization. One group (Lavitrano) was able to create transgenic mice by incubating sperm with foreign DNA and impregnating mice by in vitro fertilization, but it was not reproducible by other labs.

Current strategies in molecular medicine include antisense suppression, intracellular immunization, and virus-mediated molecular "surgery". Initial work with antisense suppression was performed with Rous sarcoma virus. Suppression of the HIV tat gene, influenza virus, and HTLV-1 have also been performed. The technology can also be used to research the function of proteins; for example, the shiverer mouse was created by microinjection of a vector carrying antisense mRNA for myelin basic protein. The transgenic mouse has less endogenous MBP and less myelin in the CNS.

Intracellular immunization is a strategy based on causing the cells to over-express a mutant viral protein, such that infectious virus cannot be synthesized when the animal is infected. This has worked in transgenic chickens expressing the avian leukosis virus envelope glycoprotein, other chickens expressing the hemagglutinin-neuraminidase gene for Newcastle Disease, mice that express human beta interferon, and other mice expressing the promoter for influenza virus.

From Founder to Strain

There are many opinions about the best way to maintain transgenic lines of mice. Some labs cross hemizygotes, and some attempt to "homozygose" their lines. The easiest way to derive homozygous animals from the founder transgenic is to do brother-sister matings of hemizygous progeny, if possible those carrying a single copy of the transgene. If the F1 is backcrossed to the founder there is a greater likelihood of inheriting the transgene at multiple sites, because the founder may be a mosaic. Homozygotes in the F2 litters is best proven by using a Southern blot in which probes for both the transgene and another gene that is conserved and constant in dosage in all the animals (i.e. EcoR1). This yields bands that can be compared either visually or densitometrically to compare the "dose" of the genes in the offspring. Mendelian inheritance would predict that if two hemizygotes are mated, then 25% of the offspring will be nontransgenic, 25% hemizygous, and 50% homozygous. It is therefore important, although laborious, to do Southern blots on all the offspring. Even then, there is a possibility for error. An animal thought to be homozygous should be mated to a nontransgenic, to see if 100% of the offspring inherit the gene. If they do not, then either the mouse was not homozygous or the gene is unstable and was lost at some time during development.

Most background strains are 129 strains, since they are source of ES cells. 129 mice are poor breeders, so it is a good idea to transfer the mutation to another background strain.{4530}

Genetic variability can come from several sources:{4530}

Genetic quality can be maintained by housing strains of different coat color in adjacent cages (to prevent confusion), using different colored cage cards, keeping proper breeding records, maintaining large foundation stocks, performance of regular genetic monitoring (i.e. annually), and cryopreservation. Repositories of genetically engineered mice include:{4530} 

Repositories assist with strain selection, and then help to ensure health quality, genetic quality, perform cryopreservation, develop models and distribute the mice or cryopreserved material to other investigators.{4530}

Speed Congenics

Strains can be generated using numerous markers (>100) to select those that are carrying more of the host genome. The best two males at each generation are selected by performance of genome scans. Backcrossing is conducted for at least 5 generations. One female is used, usually at N1, to ensure the correct Y chromosome is included.{4530}

Techniques for detecting transgenes

There are two categories of transgenes: those that have an endogenous counterpart in the recipient, and those that do not. If there is no endogenous counterpart, then either the gene transcript or its product can be easily identified in the animal. If there is also an endogenous gene, it can be difficult to identify the transgenic. One method is to introduce a small DNA tag along with the transgene that can be easily identified; however the tag may interfere with the gene. 

Often the first step in evaluating transgene expression is to look at mRNA from the transgenic animal. Many organs are sampled and homogenized in 2-mercaptoethanol or guanidine hydrochloride using a tissue homogenizer. Physical separation of the RNA is then performed by differential centrifugation through cesium chloride, phenol extraction, or differential precipitation. 

Slot-blot can be done on a large number of samples in a single experiment. RNA is applied to a nitrocellulose membrane by vacuum in a Plexiglas manifold. A radiolabeled probe is applied and detected by autoradiography. Densitometry provides a semi-quantitative estimate of the RNA in the sample. 

In a Northern blot, the size of the transcript can be estimated, which cannot be accomplished with a slot-blot. RNA is separated by electrophoresis, and then transferred to a solid membrane support. The support is then hybridized to a radioactive probe to detect the transgenic RNA.

In the Southern blot technique, the animal’s DNA is isolated, digested with a restriction enzyme such as HindIII, blotted onto nitrocellulose, and hybridized with a radiolabeled transgene-specific probe. Southern blot determines whether the transgene is present, intact, how many copies there are, and what their orientation is. Neither PCR nor slot-blot give this much information in a single analysis, partly because restriction enzymes are not used and the likelihood of false positives is higher. Slot-blot only tells whether the gene is present and the approximate copy number. PCR amplifies an internal segment of DNA and helps confirm the presence of the transgene, but is only semiquantitative.

The most sensitive way to detect transgenes is reverse transcriptase PCR (RT-PCR). A specific antisense primer is added to the RNA solution, along with reverse transcriptase to make cDNA. A second sense primer and thermostable DNA polymerase are then added, and the DNA is then denatured and re-annealed through many cycles to amplify it.

In whole tissues, in situ hybridization can be used to detect gene expression at the level of individual cells. The technique yields very high resolution when Sulfur-35-labelled probes are used to detect mRNA, and histologic section exposed to X-ray film or dipped directly in emulsion. The technique is very difficult and gives information about a single animal, but it gives the most information regarding the localization of gene expression. If the gene product is expressed in tissues, it can be detected using in situ immunohistochemical staining. 

Production of Other Transgenic Species

Rats and rabbits produce relatively large numbers of embryos, and the embryos can withstand the handling required to produce transgenics. Hamster embryos are not as suitable. Rats are excellent models for neuroanatomy and hepatocarcinogenesis, as well as mammary carcinogenesis. Most mouse mammary tumors are related to MuMTV, whereas in rats and humans, viruses are not typically involved in tumor development. The first transgenic rats were produced expressing a gene causing hypertension, which had failed to produce hypertension in mice. The first transgenic rabbits expressed a growth hormone gene. Rabbits were also the first animal model of atherosclerosis. The rabbit is the only small laboratory mammal that is susceptible to infection with HIV-1.

In contrast, the production of transgenic poultry has been very difficult. Chicken eggs cannot be microinjected; there are multiple pronuclei. Avian retroviruses have been used, but they are pathogenic to the birds and nonpathogenic viruses are not yet available. There are four methods in use to produce transgenic poultry: (1) insertion of DNA into unincubated blastoderm; (2) insertion of DNA using viral vectors (avian leukosis virus); (3) insertion of DNA using replication-deficient viral vectors; and (4) sperm-mediated gene transfer. 

Fish embryos can be microinjected with DNA; however, cytoplasmic rather than pronuclear injection is used because the pronuclei cannot be visualized. In zebrafish the following procedure is successful:

1. Use 7-18 month old fish
2. Transfer males to female tank 1-2 hr before dark cycle (sperm is called "milt")
3. Siphon bottom of tank to collect eggs 40 minutes after light cycle begins
4. Filter through nylon and rinse
5. Remove chorion with 0.25% trypsin
6. Microinject embryos
7. Transfer embryos to 10% Hanks saline
8. Embryos hatch 3 days later
9. Feed baby fish the next day, using paramecia

A major limitation to microinjection is the number of embryos that can be injected in a day. Mass transfer would improve embryo yields significantly. Electroporation involves brief electrical charges to the egg membrane, allowing it to become permeable to DNA. This has been used with loach and red crucian carp successfully. Fish sperm differ from mammalian sperm in that they are immobile in the seminal plasma, as are the eggs. However, when egg and sperm are combined in water, the fertilization process is active for a very brief period (30-60 seconds). Exogenous DNA has been encapsulated into immunoliposomes and bound to sperm, enabling sperm-mediated gene transfer in loach and common carp. Lipofection is the encapsulation of DNA in a phospholipid bilayer and binding to a cell (either egg or sperm); it has not yet been successfully performed in fish. No fish retroviruses are known which can be used to create transgenic fish.

Transgenic swine and sheep have been produced that express recombinant growth hormone. However, the genes do not result in increased growth rates unless homologous GH is the transgene, and the transgenic animals have a host of other health problems including lameness, peptic ulcers, lethargy and impaired reproductive function. Much research surrounds the production of transgenic milk-producing animals that can be used to supply proteins of interest.

Top of page

Chemical Mutagenesis

Although targeted mutagenesis ("knockout" and "knockin") are receiving a lot of attention, many molecular biologists are falling back on the older technology of creating point mutations in genes using ethylnitrosourea (ENU). 

Methods of behavioral research

Natural behavior can be observed for the presence or absence of a particular response. Four methods of data collection are used. In ad libitum scoring the animal is observed and all occurrences of a response are noted. This is a way to develop an ethogram or categorized list of responses, but the data can't be summarized for frequency or hourly rate of response. Focal animal observations record the onset and cessation of the behavior in a single animal, and are good for a large number of behaviors that have short duration. Instantaneous group scans assess things like sleeping by observing the group's behavior at pre-selected times. Data are in the form of point samples which can be extrapolated to a proportion of time spent involved in each behavior. One-zero samples record whether or not a response occurs in a set time period, such as number of scratches. This method is useless for measuring duration, but it is used to calculate probabilities. A combination of focal animal observations and group scans is often used to collect the most data in the least amount of time.{4074}  

Conditioned behavior testing places the animal in a situation in which it must learn to respond to a stimulus. In classical conditioning, reflex actions are entrained to a stimulus that would not normally elicit that reflex (like ringing a bell before food appears). Classical conditioning enables investigation of behavior that is not under conscious control, such as daily activity level that can be entrained to a flashing light. In instrumental or operant conditioning, the animal is rewarded for a correct response. Operant conditioning can be varied to make the reward contingent on a specific number of responses (fixed-ratio) or a variable number of responses (variable-ratio). These are used to test drugs for their effects on behavior. The interval between the response (i.e. pressing a lever) and the reward can also be fixed or variable; this was used to investigate the effect of lead and mercury on the squirrel monkey's ability to transition from one task to another. Acute avoidance learning involves the animal making an appropriate response to avoid an aversive stimulus, and is used to test memory retention. Passive avoidance learning requires that the subject inhibit a learned response to avoid the aversive stimulus, and is used also to test memory retention. Discrimination learning involves letting the animal make a choice between a correct response (which is reinforced) and an incorrect response (which is not reinforced). This measures the animal's ability to learn and remember antecedent-consequent relationships. Reversal learning involves training an animal to one criteria (i.e. shape) and then reversing the correct and incorrect stimuli (i.e. learning that it is the color, not shape, that indicates which is correct). One interesting tidbit of information that came from behavioral research is that all male and a third of female squirrel monkeys are red-green color blind.{4074}

Neurobehavioral assessment scales are used to study the effects of stressors on behavior, endocrine or immunologic responses. Infant assessment of primates began in the 1960s. It was learned that the amount of maternal assistance given to an infant determined the strength and persistence of behaviors associated with maintaining maternal contact. Species that have low levels of maternal assistance, such as squirrel monkeys and cotton top tamarins, have stronger, more persistent infant behaviors such as rooting, clasping, head-up orientation, and following, than high-assistance species such as rhesus. The most widely used human scale is the Brazelton Neonatal Behavioral Assessment Scale, which has been modified for primates and called the Infant Behavioral Assessment Scale (IBAS). In monkeys it is administered at 2 weeks of age. The IBAS consists of a visual orienting cluster of measurements (visual orienting and following), motor maturity cluster (head posture and righting of the head), motor activity cluster (coordination, crawling), and state control (assessment of the infant's emotional state during the test, amount of struggle, and ability to be consoled).{4074}    

IBAS was used to demonstrate that infants from chronically stressed pregnancies (caused by several social group changes) had poorer motor abilities, impaired balance, and reduced post-rotary nystagmus than those from no-stress or single-stress pregnancies.{4074} A similar testing method was used on 182 rhesus infants to show that development of hydrocephalus later (5 months to 5 years) could be predicted from the results of testing performed on days 7, 14, 21 and 30. Infants destined to become hydrocephalic showed inability to right themselves from a supine position on day 7 and higher scores on the ability to place their hands on a table after it touched the backs of their hands. However, there were only 6 hydrocephalics in the entire group, and their scores on the many parts of the IBAS were within the "normal" range. Hydrocephalics also had lower scores in the orientation cluster, in accord with visual-motor function in humans with the same condition. This most likely stems from either less mature visual systems or behavioral inattention during that portion of the test.{4095}

Rodent Activity and Behavior Testing

This table is copied from an article in Lab Animal {4605}, which took it from What's Wrong With My Mouse?

Comments? Send an email to rodgers@uky.edu