Animal & Veterinary
How Transgenics are Produced
by Susan B. Harper, D.V.M., M.S., ACLAM
FDA Veterinarian Newsletter July/August 1999 Volume XIV, No IV
For thousands of years, man has attempted to improve animal genetics by selective breeding. Targeted mating strategies are based on the presence or absence of specific traits that can be identified and transmitted to offspring. Improvements have been limited to naturally occurring events or mutations. Starting in the early 1970’s, the advent of recombinant DNA technology has introduced a variety of new techniques intended to accelerate and refine the process of genetic manipulation.
Transgenics is the science of intentionally introducing a foreign gene or genetic construct (series of genes and associated regulatory elements) into the genome of a target animal. Initial work involved a splicing technique to insert foreign genetic materials into mammalian cells maintained in culture. This in vitro work rapidly progressed into laboratory rodents, providing a more targeted and proactive approach for the establishment of new animal models for biomedical research. The results have been very successful and provide a unique and precise mechanism for the study of a variety of specific conditions or diseases with a genetic basis or influence.
The development of transgenic applications in livestock is a logical progression for this technology. Insertion of modified human gene constructs into livestock is being utilized to create "designer production animals" capable of producing useful proteins, tissues, and organs for pharmaceutical and biomedical use. Additionally, the manipulation of indigenous gene sequences has the potential to convey enhanced disease resistance and/or improve production in target animals. The primary objective in using transgenic technology in animal agriculture is to improve the quality of livestock by altering the animal’s biochemistry, hormonal balance, or harvested protein products. Scientists hope to produce animals that are larger and leaner, grow faster and are more efficient at using feed, more productive, or more resistant to disease.
Process To Create a Transgenic Animal
There are several techniques for the production of a transgenic animal and new processes are continually being developed or refined. All have the same primary objective, which is the successful integration of a functional sequence of a DNA strand (a transgene) into a chromosome within the host genome. Most of the following methods for introducing transgenes into animals have been used since the 1980’s.
Viral Vector-Mediated Transgene Introduction
The first genetically altered embryos were created using viral vectors in the early 1970’s. This technique is still in use. A viral vector (or phage) is first modified so that it will not replicate or cause disease in the target cells of the host embryo. The gene(s) of interest is incorporated into the viral genome and the virus is then used to infect an early stage embryo. The viral vector binds uniformly to the embryonic cells and acts as a vehicle to allow transfer and integration of the transgene into the host genome. Many of the experimental human gene therapy trials currently underway use basic viral vectors as a means of "gene-delivery" to susceptible cells or tissues in a very similar procedure.
An advantage to using viral vectors is that usually only a single copy of the transgene is integrated into the genome. If the viral transfection is applied to oocytes prior to fertilization, then the novel gene will be present in all cells of the resulting embryo as though it had been contributed by the maternal germline.
The major disadvantage of this system is the time and labor-intensive process to prepare the viral vector. There is also a remote possibility that the modified viral vector may revert to its original state or recombine with other pathogenic viruses.
Pronuclear-Mediated Transgene Introduction (Microinjection)
Most existing transgenic animal lines have been produced using pronuclear microinjection. This technique involves the injection of genetic material into an early-stage embryo to create what are called germ-line transgenic animals.
The gene (or genes) of interest is first identified and the nucleotides for that segment of DNA is sequenced. Frequently, there is a special segment of DNA in addition to the gene of interest, which is referred to as a promoter. The promoter is a regulatory segment of DNA located on the same chromosome as the gene of interest. It influences or controls expression of the gene. An endogenous promoter may be modified during transgene assembly in order to increase the likelihood that the gene will function in the targeted tissues of the host animal. The promoter can also be used to turn the gene on or off as needed. For example, a promoter sequence that requires a specific dietary "trigger" substance can be used to turn on genes for important hormones in animals so that the hormone is only produced when the animal is fed the appropriate trigger. A majority of the current research focuses on the understanding and development of useful promoter sequences to control transgenes and mechanisms for more precise insertion of the transgene into the recipient animal.
The prepared DNA construct (transgene and promoter) is usually replicated in a plasmid vector to produce multiple exact copies for microinjection into the pronucleus of an embryo. The injection volume is quite small (approximately 2 to 3 picoliters) and is accomplished by means of a very fine glass micropipet which is able to penetrate the cell membrane of the fertilized ovum without damage. Although many copies of the transgene construct are injected, the actual number of copies that eventually incorporate into the host genome will vary. If the transgene successfully integrates into one of the chromosomes of the pronucleus, the transferred genetic material should be present in every cell of the resulting animal and have the potential to be transmitted to future offspring.
A major disadvantage of the pronuclear microinjection system is that the rate of integration of the transgene may not be uniform between cells. Certain cell populations may not include the new DNA or may have multiple copies. Likewise, it is not always possible to specify, or target, the locus (or specific location) of integration for the transgene into the host DNA. Frequently, integration site may be a critical determinant of the transgene’s expression and function may be impaired even though the transgene is present. Similarly, if the integration of the transgene disrupts a functional DNA sequence in the host’s genome, an insertional mutation may result which interferes with the function of the existing gene.
Targeted Transgene Insertion by Homologous Recombination (Embryonic Stem Cell)
The use of homologous recombination has facilitated the development of targeted transgene insertion and, by consequence, the production of better-defined transgenic research models. The term homologous recombination refers to the exchange of DNA fragments between two DNA molecules at an identical site, which allows insertion of the transgene to be targeted to a specific location on the chromosome.
Undifferentiated embryonic stem (ES) cells have the potential to differentiate into any type of cell within the developing organism. These cells are harvested from a blastocyst stage embryo and cultured in an in vitro environment. The transgene is attached to a DNA construct that is analogous to a segment of the host DNA (except for the presence of the transgene). The new DNA-transgene is then introduced into the nuclei of ES cells in culture by means of a vector or by electroporation (the application of an electric current to enhance cell membrane permeability). As cell division occurs, the novel DNA replaces the existing segment and is incorporated into the nucleus of some of the new cells. Transgene positive cells are identified and sorted using various selection techniques, including survival selection (positive-negative selection) and polymerase chain reaction (PCR) amplification. The modified ES cells are then injected directly into a normal blastocyst embryo.
The advantage of this system is that only a single copy of the transgene is incorporated into the new cells. Also, the site of integration is highly controlled. Unfortunately, the system is very time-consuming, in that the DNA sequence of the segment of interest for the host cell must be known in order for the transgene to be constructed.
The recently much-publicized successful cloning of livestock has raised interest in the use of this technology for the production of transgenic animals. Cloning is the process of nuclear transfer, as opposed to single gene transfer, and results in the production of genetically identical animals (clones).
The nucleus of an unfertilized oocyte is removed and replaced with nuclear material harvested from a cell from an existing animal of the same species. The donor cells are frequently of epithelial origin and contain a full complement of DNA (diploid), in contrast to the unfertilized oocyte with half the total (haploid). A modified gene sequence can be introduced into the cultured donor cells prior to nuclear exchange. The nuclei of cells that successfully incorporate the transgene are identified, isolated, and inserted into recipient oocytes. Cell division is activated and the resulting embryos are implanted into the uterus of a foster recipient.
The major advantage of this system is that the generation of a large number of animals from a single donor is possible. Of course, the technology is still in the early stages and specific procedural elements are frequently being modified or evolving. Ethical issues related to the transfer of this technology into human medicine are the source of much debate.
Although these technologies are primarily restricted to research settings at the current time, it is inevitable that they will be incorporated into more traditional situations in the near future. It is very difficult to anticipate or comprehend the ultimate impact that will result. Consequently, a basic understanding and appreciation of the science involved is essential to the critical assessment of these ideas.
On March 23, 1999, the U.S. District Court for the Central District of California accepted and entered a Consent Decree of Permanent Injunction between the United States and Ivan A. Wood, doing business as Woodcrest Dairy. Woodcrest Dairy is a livestock producer located in Ontario, California.
An FDA investigation of Woodcrest Dairy’s operations revealed that since 1993, Mr. Wood caused numerous illegal drug tissue residues in livestock intended for food in spite of multiple written notifications from FDA, the U.S. Department of Agriculture (USDA), and the California Department of Food and Agriculture warning him of previous violations and the need to improve poor husbandry practices in his business.
Under the terms of the Consent Decree, Mr. Wood voluntarily agreed to be permanently restrained and enjoined from directly or indirectly introducing or causing to be introduced into interstate commerce any livestock or their tissues until the corrective actions enumerated in the decree are established and implemented. Some of these corrective actions are: (1) the implementation of adequate recordkeeping practices documenting the administration of drugs and withdrawal periods in livestock; (2) the establishment of a drug inventory and accountability system that will help to prevent future sales or distribution of animals that may contain illegal drug tissue residues; (3) the presence of a system that will prevent the administration of drug in excess of approved dosage, sale of animals before drug withdrawal period, storage of expired drugs and extra-label use of drugs; (4) the implementation of a system ensuring the segregation between medicated and unmedicated animals. In addition, Mr. Wood cannot cause the adulteration of food or drugs at any time in the future. Failure to comply with the terms of this Consent Decree may result in civil or criminal penalties.
The FDA’s Los Angeles District Office conducted the investigation that lead to this Consent Decree. The FDA/CVM’s Division of Compliance, the FDA’s Office of the Chief Counsel, and the U.S. Department of Justice’s Office of Consumer Litigation were responsible for the case processing and legal procedures.
by Tania D. Woerner, V.M.D.
March 27 & 28 of 1999, marked the first gathering of forty-five equine veterinarians from the states of Oregon, Idaho, Washington, and Maryland at the Idaho Equine Hospital in Nampa, Idaho. The group of eager participants listened to prominent equine medicine speakers Virginia Reef, DVM, Dip ACVIM; N. Ed Robinson, B. Vet. Med., Ph.D., MRCVS; Joseph Bertone, DVM, MS, Dip ACVIM; and Frank Andrews, DVM, MS, Dip ACVIM. The speakers focused on the topics of Equine Musculoskeletal Ultrasonography, Chronic Respiratory Disease, Neurologic Disease and Gastric Ulcer Disease.
The hands-on ultrasound portion of the conference was held in the Idaho Equine Hospital, a large animal medical and surgical facility. The hospital, built in 1998, has several conference rooms, a client consultation room, an operating room, two padded recovery rooms, an ultrasound room, a radiology room, and several general examination rooms with stocks. Three barns provide housing for the inpatient caseload. The hospital offers a full spectrum of medical services (endoscopy, ultrasound, radiology, etc) and surgical procedures such as laser surgery, arthroscopic surgery, and colic surgery. Six full-time veterinarians are employed by the hospital: Lionel C. Ickes, DVM; William J. Maupin, DVM; Shawn Gleason, DVM; Stuart Shoemaker, DVM, DACVS, Liz Scott, DVM; and Joseph J. Bertone, DVM, MS, DACVIM.
The conference was sponsored by Boehringer Ingelheim, Elwood, Kansas, Merial LLC, Iselin NJ, and MWI Distributors, Nampa, Idaho. Universal Medical Systems, Inc., of Bedford Hills, NY, provided the ultrasound equipment for the hands-on laboratory.
Lectures were held next to the clinic at the Idaho Center, (across the parking lot from the hospital) home of the Idaho Stampede. The rodeo club overlooking the arena, decorated with various "stampede" memorabilia provided a pleasant environment for the lectures, not to mention the exquisitely prepared breakfast and lunch served to the participants.
Overview of presentations on Chronic Respiratory Disease, Equine Musculoskeletal Ultrasonography, Neurologic Disease & Gastric Ulcer Disease
Chronic Respiratory Disease: Dr. N. Ed Robinson, is the Matilda Wilson Professor of Large Animal Clinical Sciences and directs the pulmonary laboratory at Michigan State University College of Veterinary Medicine. His research has focused on pulmonary disease in horses.
Dr. Robinson began with a review of the anatomy and physiology of the equine pulmonary system. Did you know that the horse breathes 375 gallons of air per minute? The majority of the speech was dedicated to what is known as "heaves", or chronic obstructive pulmonary disease (COPD). The now accepted terminology for this condition is Recurrent Airway Disease (RAD). Horses that are stabled and fed indoors are subjected to organic dust; composed of molds, fungal parts, rodent feces, insect parts, mite parts, and endotoxin (bacteria parts). The equine respiratory system responds to the foreign particles in three major ways: cough, bronchospasm and increased production of mucus. When these protective mechanisms overreact to the environmental stimuli, the horse is said to have "heaves". Clinical signs of heaves are a persistent cough and difficulty expiring air, often resulting in exercise intolerance.
It is estimated that 27 percent of Thoroughbreds in training in the United States have heaves. Even higher estimates of 33 percent have been reported in the United Kingdom and 54 percent in Sweden. Research has also shown that there may be a genetic component to the disease. When the condition persists, irreversible thickening of the bronchioles occurs. Dr. Robinson’s research has demonstrated that these irreversible changes may occur earlier than previously thought, within days of the initial episode of airway bronchospasm. Studies conducted at Michigan State University demonstrated that short exposures (7 hours) to an environment high in organic dust, such as a barn, may initiate the clinical signs of heaves. After a single exposure, 6-7 weeks of pasture rest is typically required for the heavey horse to recover.
Changing the environment in which the horse lives is the best way to treat heaves. Housing horses outdoors, with an open-sided shed for protection from the wind and rain is ideal. Horses that are particularly sensitive should not be fed hay (high in organic dust), but instead fed a complete feed or alfalfa pellets. If horses must be housed indoors, they should be bedded in wood shavings or shredded paper and fed a complete feed or alfalfa pellets. Dr. Robinson also believes that shipping in a trailer exposes the horse to high levels of organic dust. Studies have demonstrated that if long trailer rides (>6 hours) are planned, it is best to allow the horse to rest off the trailer for at least 6-8 hours to clear its airways of organic dust and debris. A horse will do this naturally by lowering its head or lying down.
Many times it is not feasible to house horses outdoors or feed a complete feed, and in addition, a few horses may not respond to even these measures. Under these circumstances it becomes necessary for pharmacologic management of horses with heaves. There are several classes of therapeutic pharmaceutics which are important in the treatment of heaves. The first-line treatment is steroids, but more attention is now being placed on the quaternary ammonium products (ipratropium bromide) and the b -2 adrenergics (clenbuterol, albuterol, salbuterol, etc.). The steroid that is most commonly used first in the treatment of an episode of heaves is dexamethasone, a very potent steroid that acts quickly to reduce airway inflammation and suppress the allergic immune response.
According to Dr. Robinson, human asthmatics are now treated earlier in the course of their disease with steroids to prevent the irreversible thickening of the airways. Unfortunately, horses cannot be maintained on dexamethasone for more than 7-10 days because of the potential for causing laminitis, a very serious condition of vascular compromise in the horses’ hooves causing chronic lameness and even loss of structural support of the foot. The quaternary ammonium compounds have not yet been developed for use in the horse, but research efforts are focused on these compounds to explore their potential bronchodilatory effects in the horse.
The b -2 adrenergics, in addition to causing bronchodilation, also are slightly anti-inflammatory and improve mucociliary clearance. Boehringer Ingelheim is the sponsor for Ventipulmin (clenbuterol) syrup, the first b -2 adrenergic approved by the FDA for the treatment of heaves in horses. It is administered twice-a-day at the dose of 0.5 mL per 100 pounds body weight for 3 days. If there is no improvement, the dose is increased incrementally up to 2.0 mL per 100 pounds. If there is still no improvement, the horse is considered a non-responder, and clenbuterol should be discontinued. Ventipulmin can be administered safely up to 30 days.
Boehringer Ingelheim is investigating a variety of bronchodilators and antiinflammatories to be administered using a specially designed face mask, which allows for the administration of aerosolized inhalant products. A disposable rubberized administration device that fits inside the horse's nostril is also being developed by 3M. With the invention of the face mask and the nasal delivery system, inhaled steroids such as beclomethasone and fluticasone will also be possible. Using steroids and b -2 adrenergics (bronchodilators) together has a synergistic effect in alleviating the clinical signs of heaves.
Another drug used in human asthmatics is furosemide, more commonly known as Lasix. It works by causing the release of prostaglandin E2 (a protective prostaglandin) and affecting the non-cholinergic, non-adrenergic receptors in the airways resulting in bronchodilation. The overall message is that effective pharmacologic agents are being developed for treating the horse with heaves and that the inhaled treatments are becoming more practical for use in the horse.
Equine Musculoskeletal Ultrasonography with Hands On Laboratory: Dr. Virginia Reef is the Director of Large Animal Cardiology and Diagnostic Ultrasonography and Chief of Sports Medicine and Imaging at the University of Pennsylvania's New Bolton Center. She is a world renowned expert in ultrasonographic imaging.
Dr. Reef’s lecture focused on the diagnosis and treatment of the "bowed tendon". A "bowed tendon" is a common injury of racehorses and nearly always occurs in the front legs involving the superficial digital flexor tendon. A bowed tendon can be a career-ending injury for a racehorse. The clinical signs of a bowed tendon are swelling, lameness, heat and pain on palpation. These clinical signs are not consistent. In fact, only 50 percent of horses with tendon damage exhibit lameness. In addition, inflammation of the tissues surrounding the tendon may look very similar to a bowed tendon. The use of diagnostic ultrasonography has revolutionized the diagnosis of tendon injury and allows for the differentiation of tendon damage from uncomplicated soft tissue swelling surrounding the tendon. Dr. Reef and Dr. Ronald Genovese, a specialist in equine distal limb ultrasound, have developed objective methods of quantifying tendon injury using ultrasonography. These objective measurements are then used to monitor tendon healing and permit the tailoring of rehabilitation to the individual horse.
Traditional treatments of "bowed" tendons consist of puncturing the tendon multiple times with a needle or other sharp instrument (tendon splitting), cutting the fetlock annular ligament which restricts the movement of the flexor tendon and cutting the superior check ligament, which anchors the superficial flexor tendon to the radius in the forearm of the horse. None of these treatments, including just resting the horse, have been proven to be effective and recurrence of the same injury is common. In 1998, Boehringer Ingelheim, gained FDA approval for BAPTEN (beta-aminoproprionitrile fumarate), a substance which blocks lysyl oxidase, the enzyme responsible for collagen cross-linking. Injection of BAPTEN into the lesion in the tendon is administered approximately one to three months after the initial tendon injury. This treatment is coupled with a controlled exercise program. At four months post-treatment it became apparent that there was a better sonographic quality of tendon repair (i.e., more parallel fiber alignment) in the BAPTEN treated horses as compared to the placebo treated. Generally, 6 months of low intensity exercise following treatment is necessary to ensure that adequate time has passed for collagen cross-linking to occur and for the tendon to regain its strength.
The ultrasound laboratory was held at the Idaho Equine Hospital. The veterinary staff served as instructors and demonstrated ultrasound techniques using two different ultrasound systems (Sonovet 600 & Ausonics Impact VFI). Participants were trained to evaluate the equine umbilicus, abdomen, chest, eye, tendons and reproductive tract of the mare.
Equine Neurology: Dr. Joseph Bertone is a member of the Idaho Equine Hospital. He is board certified in Large Animal Medicine and has published and presented information in the areas of shock, physiology and neurology. He is a renowned speaker in the area of equine internal medicine.
Dr. Bertone presented an overview of equine neurology and the diseases associated with the neurologic system. Equine Protozoal Myeloencephalitis (EPM) a devastating neurologic disease caused by a protozoan organism (Sarcocystis neurona) was discussed in depth. EPM affects all breeds of horses, but is more commonly reported in Standardbreds and Thoroughbreds. There is no age restriction; however, the disease appears to affect younger horses (average age is 4 years), typically when competing or racing. The disease often presents as an asymmetrical gait deficit, which may be confused with lameness. Dr. Bertone discussed the neurologic examination and special tests that can be conducted to differentiate EPM from other types of neurologic disease and lameness. EPM is treatable, but often times not curable as the residual damage in the central nervous system is often irreversible. Standard treatment is long-term administration of a combination of a sulfonamide antimicrobial drug and pyrimethamine. Although this combination is not approved for treatment of EPM, extensive research is underway to determine the effectiveness of this combination and several other pharmacologic agents such as diclazuril (Clinacox), toltrazuril (Baycox) and nitazoxanide.
Dr. Bertone also discussed the procedure for collection of cerebrospinal fluid (CSF), necessary to diagnose EPM. Collection of CSF requires a 8-inch needle, proper restraint and sedation and correct anatomic placement of the needle. CSF fluid is subjected to a test named the Western Immunoblot test to determine if antibodies to the protozoan are present. Detection of antibodies in the CSF is somewhat problematic in that blood contamination, either from the introduction of the needle during the procedure, or from trauma to the spinal cord or brain, may result in a false positive test result. It has been shown that up to 45 percent of the normal horse population has been exposed to the organism and carries antibodies in the blood. Only those that have antibodies in the blood and in the CSF are considered to have active disease. Two tests, the albumin quotient and the IgG index have been developed to help rule out the blood contamination as the cause for a positive test result. It is now known that a very small amount of blood contamination may result in a positive test. Research is underway in this area to refine the Western Immunoblot test. Dr. Bertone also presented videotapes of neurologic horses and discussed the neurologic examination as the horse was being examined.
Equine Gastric Ulcer Disease (EGUD): Dr. Frank Andrews is a Professor and Section Chief of Large Animal Medicine, University of Tennessee College of Veterinary Medicine. His research focus is gastric physiology and gastric ulcer disease in horses. He is well published and considered an international expert on this subject.
Dr. Andrews reviewed the anatomy and physiology of the equine stomach. The equine stomach is separated into two portions. The upper portion of the stomach is lined by squamous epithelium. The lower portion is lined by glandular epithelium. Gastric ulcers in adult horses are most commonly found in the squamous portion of the equine stomach. The squamous portion is more sensitive to acid than the glandular portion because the glandular portion is able to secrete a protective layer of mucus. Clinical signs of gastric ulcers in horses are variable and may include mild signs of abdominal discomfort (colic), poor appetite, weight loss, and poor performance. A definitive diagnosis can only be made by gastric endoscopy, which requires a 3 meter endoscope. The horse must be fasted at least 12 hours prior to the procedure. The stomach is insufflated with air and the contents are aspirated. Most horses are mildly sedated for this procedure.
Dr. Andrew’s research has shown that the pH of the gastric fluid is dependent on the diet of the horse. The horse’s digestive tract is best suited to continuous grazing, not intermittent feeding of large meals. Domestication of the horse has imposed our eating habits upon the horse, some of which may have deleterious effects. For example, it has been shown that large concentrate grain meals result in more acidic gastric contents. The consumption of hay, especially alfalfa hay, has the ability to buffer some of the acid that is produced. Studies have shown that subjecting a horse to a strenuous exercise program (race training) may induce ulcers. Modifying the feeding schedule and environment of a horse may allow for management of gastric ulcers. Smaller grain concentrate meals should be fed along with better quality and continuously accessible hay. Ideally horses should be housed outdoors and have continuous grazing. Once again the demands of a performance horse may make this type of management difficult and pharmacologic agents should be considered.
A visit to the anti-ulcer section of your local pharmacy reveals the variety of over-the-counter medications available to humans suffering from gastric ulcers. In contrast, the first gastric ulcer medication (GastroGard/omeprazole) was developed by Merial LLC and FDA approved in 1998. Omeprazole acts by irreversibly binding to the enzyme responsible for the production of acid from the parietal cells in the stomach. It is approved for human prescription use as Prilosec. GastroGard is available as an oral paste and is to be administered once-a-day for a period of 28 days, after which the dose is halved and administered for at least four more weeks. Dr. Andrews conducted clinical trials with omeprazole and demonstrated by gastric endoscopy that omeprazole was 77 percent effective in healing ulcers after a period of 28 days and that in 84 percent of horses, recurrence of ulcers could be prevented by continued administration of omeprazole for an additional four weeks of treatment. Other medications for the treatment of gastric ulcer disease such as cimetidine (Tagamet), ranitidine (Zantac) and sucralfate (Carafate) are not approved for use in the horse and have not been subjected to the well-controlled studies necessary to confidently predict effectiveness. Following the lecture, Dr. Andrews showed videotapes of gastric examinations conducted during the studies. The videotapes clearly showed the ulcers prior to treatment and the resolution of ulcers following treatment.