The sophistication of patient monitoring required varies with the species and the procedure, but during protocol review, the IACUC should expect evidence of the following:
a pre-surgical assessment;
adequate monitoring of depth of anesthesia and animal homeostasis during the surgical procedure;
support such as fluid supplementation, external heat or ventilation;
monitoring and support during anesthetic recovery ; and
post-surgical monitoring details, (e.g., what will be done and how often, who will be responsible, and the name and phone number of the individual to contact in the case of post-surgical complications).
Recordkeeping Recordkeeping is an essential component of peri-operative care. For major surgical procedures on non-rodent mammals, an intra-operative anesthetic monitoring record should be kept and included with the surgeon’s report as part of the animal’s records. This record should be available to the personnel providing post-operative care. Post-operative records, at a minimum, should reflect that the animal was observed until it was extubated and had recovered the ability to stand. These should be supplemented by records evaluating the animal’s recovery, administration of analgesics and antibiotics, basic vital signs, monitoring for infection, wound care, and other medical observations.
Occupational Health and Safety Surgical situations can present certain occupational health and safety risks related to:
use of inhalation anesthetics,
use of certain species or a species under certain circumstances (e.g., pregnant sheep), or
use of certain devices (e.g., lasers).
If the circumstances warrant it, the IACUC should consult with the appli-cable biosafety personnel.
References Brown, M.J., P.T. Pearson, and F.N. Tomson. 1993. Guidelines for animal surgery in research and teaching. AJVR (54), 9:1544-1559.
National Agriculture Library, 1994. Essentials for Animal Research: Principles of Aseptic Technique. 41-50.
National Agriculture Library, 1994. Essentials for Animal Research: Perioperative Care. 51-66.
Mangram, A.J., T. C. Horan, M.L. Pearson, L.C. Silver, and W.R. Jarvis, 1999. Guideline for Prevention of Surgical Site Infection, 1999. Inf Cont & Hosp Epi (20), 4:250-278.
Academy of Surgical Research. 1989. Guidelines for training in surgical research in animals. J Invest Surg 2:263-268.
A spontaneous mutation is a naturally occurring heritable alteration in the genetic code. Spontaneous mutations have been observed in virtually all species. An induced mutation is a man-made alteration in the genetic code. Induced mutant is a generic term including transgenic and targeted mutations that are created to study over-expression or under-expression of a specific gene. The altered gene must be predictably transmitted to offspring for a spontaneous or an induced mutation to be useful in research. To date, the majority of induced mutations have been made in laboratory mice of the genus Mus or laboratory rats of genus Rattus. Although mice are used as examples in the following discussion, the general considerations are applicable to induced mutants of any species.
Transgenic refers to insertion of exogenous DNA (deoxyribonucleic acid) into cells. Typically, cDNA (complimentary deoxyribonucleic acid) made from specific mRNA (messenger ribonucleic acid) is inserted into cells using microinjection, electroporation or certain nonpathogenic viruses. (Electroporation is the brief application of an electric field to a cell to increase permeability of the cell membrane for purposes of introducing drugs or genes into the cell.) Each of these methods has been used to insert new DNA into the pronucleus of a fertilized mouse egg and to create transgenic mice. The manipulated fertilized eggs may or may not be cultured in vitro for one to three days before they are surgically implanted into the oviducts or uterus of pseudopregnant female mice. The inserted DNA incorporates in chromosomes of a percentage of embryos developing from the microinjected eggs. The DNA incorporates at different genetic locations and a different number of copies of the DNA may incorporate in different embryos. Thus, each embryo has the potential to become a unique transgenic mouse even though the same quantity and type of DNA was injected into genetically identical fertilized eggs. All manipulated, fertilized eggs do not become live born transgenic mice. Losses occur at every step from injection through gestation and delivery.
Mice can carry transgenes, but unless the cDNA is incorporated into germ cells, the mouse is unable to transmit the transgene to its offspring. A mouse that passes the transgene to the descendants is called a ‘founder. Thus, many fertilized eggs have to be injected to obtain a few transgenic mice, and only a few of these transgenic mice will be ‘founders’ of this transgenic line.
Targeted mutation refers to a process whereby a specific gene is made nonfunctional (‘knocked-out’) or less frequently made functional (‘knocked-in’). Creation of a targeted mutation requires several steps in the laboratory. The specific gene is identified, cloned and manipulated to make it nonfunctional (‘knocked-out’). The manipulated gene is attached to another DNA sequence called a promoter and introduced into embryonic stem (ES) cells by electrical or chemical methods. These ES cells are cultured in special media that permits identification of ES cells incorporating the manipulated gene. ES cells incorporating the manipulated gene are injected into an early embryo (blastocyst). The ES cell injected blastocysts are surgically implanted into the uterus of pseudopregnant female mice. Some injected blastocysts develop into viable embryos and gene deficient ‘knock-out’ mice
are born. Many blastocysts have to be injected to obtain a few new ‘knock-out’ mice, and only a few of the new ‘knock-out’ mice will incorporate the ‘knocked-out’ gene in their germ cells and become ‘founders’.
If a project uses a spontaneous or induced mutant model and the mutant animal can be purchased from a resource or commercial colony, review of this project is similar to review of any other project. If a project uses an induced mutant model and only breeders are available from the source, review of this project is similar to review of any other breeding colony. In either case, the IACUC should determine if the mutant gene will result in a severely debilitating phenotype, if anything can or will be done to ameliorate such phenotype, and what endpoints will be used to determine when a mutant animal will be euthanized. Simple husbandry measures can modify the severity of some mutant phenotypes. For example, ground feed or moist feed can extend life and improve growth of mutants with missing or malformed teeth. Food and water on the bottom of the cage may be easier for mutant rodents with neuromuscular abnormalities to access than food in a traditional feeder built into a cage lid. Extra bedding helps dwarf mice reach food and water. Extra bedding helps absorb urine produced by diabetic mice or other mice that excrete large quantities of urine. A normal cage mate, a solid bottom cage with extra bedding, or a slight increase in room temperature can benefit mutant rodents that have problems maintaining body temperature (Beamer, 1986).
When an investigator prepares a proposal that includes development of a new mutant model, information about clinical abnormalities associated with the phenotype, special husbandry requirements, etc. will not be available. However, the investigator should include general criteria for euthanasia if a severe debilitating phenotype develops, and provide the IACUC with this information when the new mutant has been developed or at the next annual review.
The standard of ‘normal’ for a mutant animal may or may not be the same as for a non-mutant animal. If the mutant phenotype does not impact clini-cal well-being of the animal, the same standard of ‘normal’ can be used for mutant and non-mutant animal. In the mouse, brown (gene symbol Tyr) and short ear (Bmp5) are examples of spontaneous mutations that
produce no observable, clinical impact on the well-being of the mouse. If the mutant phenotype has minimal impact on the well-being of the animal, the standard of ‘normal’ can be similar for mutant and non-mutant animal. Hypogondal (Gnhr) and ‘little’ (Ghrhr) are examples of spon-taneous mutations with minimal impact on well being of the mouse. Homozygous hypogondal mice are normal in all ways except for small, nonfunctional gonads. Homozygous ‘little’ mice are smaller than non-mutant littermates. Growth hormone transgenic mice tend to have larger body size than normal, but are otherwise clinically normal with the exception of reduced fertility.
In the case of mutants where phenotype involves clinical abnormalities, the standard for ’normal’ may have to be modified to encompass the expected phenotype. For example, 4 to 5 week old homozygous dystrophic mice (Lama) have difficulty abducting hindlegs and have an abnormal gait. As these mice age, muscular weakness progresses in hindlegs and eventually extends to involve all skeletal muscles. The standard for ‘normal’ for homozygous dystrophic mice must include difficulty abducting hindlegs and an abnormal gait. Adenopolyposis coli ‘knock-out’ mutant mice (Apc) are clinically normal until the intestinal polyps develop, after which time the mice become anemic and lose weight. Experimental endpoints for these latter and similar mutant models should focus on (1) ability of the mutant to access feed and water, (2) response of the mutant to stimuli, and (3) general condition of the mutant, (i.e., is the mutant excessively thin, showing progressive weight loss or hunched posture?).
Many institutions have a centralized induced mutant facility that receives the genetic material from investigators and performs the manipulations to develop ‘founder’ transgenic or ‘knock-out’ mice. The ‘founder’ mice are returned to the investigator who undertakes breeding to expand the line. Review of the centralized induced mutant facility should focus on personnel qualifications, animal related practices such as aseptic surgery, and average number of mice required to produce ‘founders’ for a single DNA construct, recognizing, however, that the number of mice required is a very rough estimate because of differences in responses of different strains or stocks of mice, variations in success rate for different DNA constructs, and subtle or less subtle uncontrollable environmental changes.
In many non-mutant model experiments, an investigator can accurately estimate the exact number of animals required to test a hypothesis. However, when creating an induced mutant, there are major variables that make it difficult to accurately estimate the number of required animals, including:
differences in percent successful microinjections of pronuclei or suc-cessful incorporations of altered gene into ES cells;
differences in percent successful incorporation of exogenous DNA or altered gene into germ cells of induced mutant mice.
Different strains of mice vary in their responses to each of these manipula-tions. Different genes (‘constructs’) vary in the ease with which they insert as a transgene or are ‘knocked-out’. These variables remain even when the same skilled people perform each manipulation.
References Beamer, W. “Use of Mutant Mice in Biological Research.” 12/8/89, SCAW Conference: Guidelines for the Well-being of Rodents in Research, Proceedings edited by H. Guttmann.
Gordon, J.W. 1990. Transgenic animals. Lab Animal 19(3):27-30.
Hogan, B., F. Constantini and L. Lacey. 1986. Manipulating the Mouse Embryo: A Laboratory Manual. Cold Springs Harbor Laboratory Press. Cold Springs Harbor, NY.
Kovacs, M.S., L. Lowe and M.R. Kuehn. 1993. Use of superovulated mice as embryo donors for ES cell injected chimeras. Lab Anim Sci 43:91-93.
Mobraaten, L.E. 1981. The Jackson Laboratory Genetics Stocks Resource Repository. In Frozen Storage of Laboratory Animals. Zeilmaker, GD (ed). Gustav, Fisher, Verlag. New York. pp. 165-1177.
Wilson E.D., and M.X. Zarrow. 1962. Comparison of superovulation in the immature mouse and rat. J Reprod Fert 3:148-158.
Zarrow, M.Z. and E.D. Wilson. 1961. The influence of age on superovulation in the immature rat and mouse. Endocrinology. 69:851-855.