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Perspectives for Molecular Genetics Research and Application in Poultry1
Perspectives for Molecular Genetics Research and Application in Poultry 1 R. N. SHOFFNER Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108 (Received for publication July 31, 1985)
1986 Poultry Science 65:1489-1496 INTRODUCTION Molecular genetic t e c h n o l o g y is n o w an integral p a r t of t h e inquiry into biological functions involved in inheritance, cytogenetics, i m m u n o l o g y , endocrinology, nutrition, behavior, r e p r o d u c t i o n , and animal health. C o m m e r c i a l applications already include t h e p r o d u c t i o n of pharmaceuticals, f e r m e n t a t i o n products, and vaccines. A considerable p o r t i o n of current f u n d a m e n t a l biological research reduces t o genetics in t h e sense t h a t d e o x y ribonucleic acid (DNA) and ribonucleic acid ( R N A ) nucleotide sequences have b e e n identified for a wide variety of biological c o m p o u n d s including globins, chick a l b u m e n , platelet growth factor, specific i m m u n o p e p t i d e s , g r o w t h h o r m o n e , insulin, interferon, and o t h e r comp o u n d s . Even t h e c o m p l e t e g e n o m e s of s o m e microorganisms have b e e n characterized. Currently, t h e enthusiasm for genetic engineering in p o u l t r y is t h e p o t e n t i a l to use gene transfer t e c h n o l o g y for genetic improve-
m e n t . However, we m u s t recognize t h a t molecular genetics is n o t a panacea or a r e p l a c e m e n t of conventional p o u l t r y breeding t e c h n o l o g y . It is a marvelous t o o l for uncovering new knowledge a n d adds t o our ability t o advance genetic i m p r o v e m e n t . Molecular genetic t e c h n o l o g y involves t h e m a n i p u l a t i o n of genes in vitro w i t h r e c o m b i n a n t D N A m e t h o d o l o g y . T h e gene r e p l a c e m e n t p r o c e d u r e substitutes a gene sequence of cloned r D N A i n t o c h r o m o s o m a l D N A of a cell or organism, either b y first removing c h r o m o s o m a l D N A and replacing it b y r e c o m b i n a n t D N A (rDNA), or merely adding in t h e extra rDNA t o create novel g e n o t y p e s . Mutagenesis is a n o t h e r manipulative m e t h o d w h e r e b y t h e gene sequence is altered in vitro, t h e n followed b y r e i n t r o d u c t i o n into t h e cell or organism — in effect, t h e creation and insertion of a new gene. A t h i r d system for altering g e n o t y p e s is t h e fusion gene t e c h n i q u e accomplished b y fusing two gene sequences or p o r t i o n s of t w o genes into a single gene t h a t has altered transcription capability.
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Paper No. 14729 in the Scientific Journal Series of the Minnesota Agricultural Experiment Station. 1489
BENEFITS OTHER THAN DIRECT GENE TRANSFER A n i m p o r t a n t area of genetic engineering
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ABSTRACT Genetic engineering of microrganisms to produce more efficient vaccines, immunizing polypeptides, monoclonal antibodies, microbes with enhanced biodegradable capabilities, and pharmaceuticals have potential usefulness for poultry producers and researchers. Molecular technology can be used for chromosome mapping. Application of gene transfer technology to poultry requires an integrated program, including molecular genetics expertise, cell biology-embryology knowledge, and breeding capabilities. A successful gene transfer in poultry promises an increase in genetic variability. A gene theoretically can be inserted directly into selected stocks without extraneous genetic material. There is the exciting potential for inserting genes from other species. The recombinant deoxyribonucleic acid (DNA) technology for isolating and cloning a gene sequence can be readily applied to poultry. Cloned DNA for a desired gene sequence requires a delivery system to get the sequence into the germ line. A retrovirus vector that includes the sequence appears to be the system of choice for oviparous species at this time. Germ line target cells are primordial germ cells, spermatozoa, ova, and early embryonic cells that will give rise to gonads that have a chromosome with the inserted sequence. Evaluation of gene transfers at the molecular level will determine if insertion is accomplished, as well as the number and location. A major portion of a molecular genetics program will include breeding evaluation for expression, stability, and usefulness, including all the conventional procedures for multiplication, selection and structuring of populations for crossing, and field testing. (Key words.- poultry, molecular genetics, recombinant deoxyribonucleic acid, delivery vectors, antisense ribonucleic acid, breeding application)
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On the other hand, the benefits for research application have considerable promise. Gene cloning is part of basic inquiry into physiological function and is becoming almost routine, and, when applied to poultry, will greatly increase our understanding of the fundamental biology of birds. Currently, securing pituitary hormones is not only a tedious process, but only minute amounts are obtained from processing immense numbers of chicken or turkey pituitaries. Cloning the gonad-stimulating hormone (GSH) gene, for example, inserting it into bacteria or cells in culture would make it possible to produce gram quantities of this or similar products. GENE MAPPING Chromosome Location. The chicken gene map has about 12 linkage groups and only a few are assigned to specific chromosomes. The major portion of the genes in the current linkage groups are those for morphological traits. Assignment to chromosomes by conventional cytogenetic methods, somatic cell hybrids, or rearrangement marker chromosome methods has not been very fruitful
because of high recombination frequency. Molecular technology utilizing radioactive marker probes, avidin-biotin probes, and similar methods now make it possible to identify single copy DNA on in situ treated chromosome preparations or to use restriction fragment length polymorphisms (RFLP) in gene-mapping studies. The gene map for chickens and other domestic poultry is expected to expand rapidly with fairly precise chromosomal locations. At the outset, the gene map will consist primarily of traits recognizable in electrophoretic gels and similar techniques, and, in time, these traits will be linked to morphological linkage groups and possibly major genes or blocks of genes influencing quantitative traits. Marker-Assisted Selection. Single genes may be only a few kilobases (kb) in length, but flanking RFLP sequences could multiply the recognizable region several times, thus increasing the number of labeled hybridizing probes as well as the receptive recognition sites on the chromosome. Morris Soller (Soller and Beckman, 1986) proposes to take advantage of the RFLP technology to identify genes or regions on the chromosome for marker-assisted selection of quantitative traits. Sex Probe. Vent sexing, plumage colors, and feather sexing at hatching is routine, because sexual dimorphism of domestic poultry makes it relatively easy to distinguish sex. Sexual dimorphism at all ages is absent or vague species, zoological specimens, and pets. The W chromosome of the heterogametic females can be recognized in karyotype preparations especially if assisted by the cytochemical differentiation C-banding technique. Determination of sex in very early embryos prior to gonadal differentiation can also be accomplished by cytochemical differentiation methods, if chromosomal preparations are available. Even though the cytogenetic techniques are workable, the identification of a W chromosome sequence that can be recognized with a specific probe would be exceedingly convenient for determining sex of cells from all types of specimens.
SOMATIC CELL TRANSPLANTS This system is designed for gene therapy, whereby genetically defective cells are made "normal" by insertion of the nondefective alleles into cells in culture and then transplanted back into the organ where the defective
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that can benefit the poultry industry involves the restructuring of microorganisms to create novel genomes with improved immunization capability. Several strategies have been used, or proposed, to modify viral genomes for vaccines or as vectors for genes that specify antigens for prevention of infectious diseases. A vaccinia virus, carrying a foreign gene sequence with specific immunizing properties, could conceivably be a more efficient vaccine for those diseases of poultry where only killed organisms are now safe to use. The identification of specific polypeptide sequences with immunizing capacity against bacteria and protozoa may lead to products protecting poultry from infection by these organisms. Reconstruction of microbes with enhanced biodegradable capacities, such as those degrading oil and halogenated compounds, may be useful in poultry waste lagoons and methane gas production systems. Pharmaceutical products such as monoclonal antibody for poultry health may have limited application, because the small individual worth may limit the use of costly therapeutic application. The value of injectable compounds such as growth hormone will depend upon the trade-off between costs of compound and injection vs. gains in performance.
SYMPOSIUM: MOLECULAR APPROACHES TO POULTRY BREEDING
GENE TRANSFER IN POULTRY To take advantage of the opportunities of molecular biology in poultry research, and especially gene transfer technology, one has to bring together an integrated program involving the expertise of molecularly trained personnel, cell biology-embryology knowhow, and, finally, but not least, breeding and genetics for evaluation and propagation. This can be accomplished in one or several laboratories, but the essential ingredients have to come together for total application. Potential Advantages. 1. Genetic variability would be increased and so would the possibility of an advantageous genetic change. 2. A gene could be inserted directly into selected, wellcharacterized stocks such as grandparent lines, inbred lines, and purebred stocks. The tedious and time-consuming efforts of many backcrosses required in the conventional gene transfer would be avoided. 3. The possibility of overcoming fertility barriers and inserting exotic genes from another species has considerable interest. 4. Unfavorable linkage situations could be broken through rearrangement and reinsertion. 5. The chromosome map would be increased as it is important to locate the original gene locus as well as that of an "add in" gene. Disadvantages. Molecular gene transfer application research is a high-cost, high-risk venture. Few poultry research units have the facilities, equipment, or personnel necessary to pursue molecular technology, and it requires considerable investment just to begin a program. There is no guarantee that gene transfer will result in dramatic genetic improvement of poultry. The frequency of failures may far
exceed successes. There is no doubt that structural genes will be inserted into the genome, although there are several technical problems to be overcome before this process becomes routine. At this point in time, identification of useful genes has to be accomplished, as well as learning about gene regulation in the complicated feedback systems of the chicken. However, I consider these to be difficult technical problems that will be solved in time, rather than complete barriers to useful gene transfers. Possible Biohazards. The probability is exceedingly low that a transformed chicken will endanger either other organisms or the environment, because they are highly contained and can be readily destroyed if necessary. Modern highly selected strains of domestic chickens, turkeys, ducks, and geese have lost a portion of their genetic adaptability to survive in the "wild." Furthermore, the inserted gene may make them even less adaptive for survival without man's husbandry. Perhaps the greatest danger could be loss of germplasm. Suppose, for example, that a very favorable genetic transfer is made into an egg production stock, and all other stocks were discarded. Subsequently, it was found the transformed birds were extremely susceptible to a particular infection, and alternative germ plasm is unobtainable. This possibility seems exceedingly remote, because it is a well-recognized problem and will be guarded against. Release of genetically engineered organisms into the environment is a concern, and precautionary guidelines have been instituted and rigorously followed to control possible problems with engineered laboratory strains of microorganisms. Restructured RNA and DNA viruses, as vectors capable of delivering genes and at the same time being capable of selfperpetuation and spread, will require careful monitoring. However, vectors have been, and future ones can be, restructured through genetic engineering, so that they are incapable of reproduction or causing infection. Jones (1985), discusses some possible situations that will require evaluation for product safety in transfected food animals. When the host's genome is altered by a gene insert that acts independently of the recipients genome, it gives an exogenous expression that may be desirable or undesirable depending upon its end product. A viral vector that retains ability to produce infectious virus, or an insert that gives a car-
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cells have been killed or inactivated in order that the transplant cells can grow and proliferate. The practical advantages of this system is to get around the usual immunological complications of transplants or into regions where organs cannot be removed and replaced. Some success has been achieved with bone marrow cells in leukemic mice, and there is considerable interest for application in human medicine. Because of the limited value of the individual chicken, even if this technology is perfected, it would probably be of little value to the poultry industry. A more feasible alternative would be a gene transfer to prevent infection, such as a resistance gene that acts endogenously to insure healthy birds.
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cinogenic product, would be extremely undesirable. Alternatively, the exogenous gene could produce a desirable effect by improving the animal's well-being. If the replicated inserts come under the control of the host's genome, the expression product is considered as endogenous. The resulting expression may be either favorable or unfavorable depending upon the nature of the product coded for by the insert.
Recombinant Deoxyribonucleic Acid Technology. Step Number one is to decide on a gene with potential value such as growth hormone or similar compound. The preference would be one with a short uncomplicated sequence, as it would be easier to identify in a genomic library, and insertion into a plasmid vector would be less complicated with a greater probability for chromosome insertion. The next maneuver is to identify and isolate the gene sequence of choice. The method of least trouble is to beg one from another laboratory. If a homologous, or nearly homologous, sequence is available from another species, for example, bovine growth hormone, the isolation of the chicken growth hormone is simplified. If these alternatives are not available, one has to start from scratch, use restriction enzymes to cleave the DNA, form a genomic library, and go through the tedious search for the desired sequence using probes designed to be complementary to a known protein sequence, or through antibody probing of an expression library. The next step is to isolate the desired sequence through the applications of recombinant DNA (rDNA) technology for multiplication of gene copies. Sequences are recombined into the chromosome of a plasmid such as pBR322, a phage, or a cosmid. Cosmids are plasmid-phage complexes usually able to hold a larger sequence than plasmids. Both plasmids and phage live and multiply in bacteria, and the laboratory strains of E. coli are usually the organisms of choice for amplification. Clones of bacteria, each with a different insertion, are grown and tested for the presence of the plasmids with the desired sequence. Once isolated, the plasmids with the gene are amplified in E. coli cultures for producing many gene copies.
Target Cells. Totipotent cells are those whose chromosomal complement will eventually be in some or all subsequent cells of the organism. The target cells of importance are the gonadal cells, a) The primordial germ cells (PGC) that arise early in embryonic development as receptors of transferred genes would be expected to be progenitors of cells producing gametes carrying the transferred gene. A gonad with transplanted PGC, carrying gene sequence inserts, theoretically would produce gametes with the transferred gene. Shuman (1983) developed a successful transplant technique, but donor chromosomally marked gametes were not identified in subsequent test matings. Consequently, while this route is an appealing one for gene transfer and other manipulations, several technical problems will have to be mastered before this system becomes useful, b) Gametes, either sperm or egg, with an inserted gene sequence, would contribute that segment to all subsequent cells in the zygote. For sperm to be efficient vectors, some method of mass treatment would have to be devised. Some thought and effort has been given to developing a viral vector (retrovirus construct for example) that would attach to sperm, thereby be transported into the egg, and thus have an opportunity for the vector to be incorporated into a chromosome of the subsequent zygote. The avian ovum is located on an extremely large yolk, and, upon ovulation, the ovum is picked up almost immediately by the infundibulum where fertilization occurs shortly before the ovum begins its journey down the oviduct to complete egg formation. Consequently, manipulation of the ovum is difficult for injection or similar procedures, c) Incorporation of calcium phosphate precipitated rDNA into the cell nucleus results in transfection of donor rDNA into the chromosomal DNA of the host cell. The newly laid fertile egg has an embryo that has developed to the blastocoele stage. At this stage or shortly thereafter, the embryo is presumed to contain inner cell mass (ICM) cells that are totipotent or at least pluripotent so that rDNA transfection of these cells may be expected to have subsequent gonadal cells carrying the transferred gene. Because embryos of this stage are readily available, there is considerable attraction for exploring this route for rDNA transfection.
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OPERATIONAL STEPS IN GENE TRANSFER APPLIED TO POULTRY BREEDING
DEVELOPING A DELIVERY SYSTEM FOR INSERTION INTO THE CHICKEN GENOME
SYMPOSIUM: MOLECULAR APPROACHES TO POULTRY BREEDING
EVALUATION FOR EXPRESSION AND FUNCTIONALITY Determination of Insertion by Molecular Technology. Following gene transfer attempts, one wishes to know if the transfer was accomplished. A successful insert into a chromosome is expected to be transmitted to progeny. The DNA secured from tissue or nuclei of red blood cells of chicks can be probed by several means utilizing dot-blot electrophoresis, autoradiographic labeled DNA (same sequence as inserted), antibiotin antibody fluoresence, or other methods to determine if the intended inserted sequence is present in the chromosomal DNA. There are reasons for making the DNA determinations besides that of merely detecting presence of the insert. The number of inserted copies may vary in number as well as chromosomal location, which, at this point, is assumed to be random. None, one, or several inserts may be expressed. Location of inserts by hybridization probes to the chromosomal regions, especially in relation to the indigenous genes, is important. Some interesting ratios
could occur if an inserted gene expresses as well as the indigenous locus. In addition to segregation ratios in expression, there would also be an independent assortment expression situation if the insert is on a different chromosome or on the same chromosome in a different locus. Dosage effects may also be involved in expression. Poultry Breeding Technology. Let us suppose that a desirable gene has been transferred and is expressed in one of the progeny. To start, we have one transfected individual that is heterozygous as insertion is expected in only one of the pair of homologus chromosomes. Dominant expression would be recognized immediately, while recessiveness requires further matings for expression. From now on in the application process, we have to depend upon sexual propagation to multiply the newly created genome with its heterozygous insert. Only 50% of the progeny will carry the new gene so all the appropriate matings, recording, and selection of parents will follow conventional breeding procedures. In successive generations, we will need to observe for stability. Does the new gene just fade away? Does it act like a transposon (jumping gene), moving from chromosome to chromosome and changing expression as it changes location? What will be the effect on other traits? Will the energy required for increased expression detract from other traits? Is the inserted gene an additional load on an already established genome? Other types of interaction effects are possible. If the new gene passes all the desirability tests, it has to be incorporated into a selected stock. Hopefully, the insert will be made into a select stock so that the transferred gene is already where it is wanted. Test crosses, field tests, and all the usual breeding work necessary for evaluating a commercial product will be required. It will take some time before the "wonder" gene appears in millions of chickens. RECOMBINANT RIBONUCLEIC ACID The technology to recombine RNA fragments has lead to what is called ANTISfiNSE RNA. The RNA is made double stranded in opposing directions and suppresses translation of RNA by forming RNA/RNA hybrids. The activity appears to be specific, as Izant and Weintraub (1985) found that antisense Herpes Simplex virus-thymidine kinase (HSV-TK) inhibits HSV-TK production but not chicken
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Delivery Systems, a) Common methods of transfecting rDNA into the nucleus are microinjection, calcium phosphate precipitate with enhancement, and liposomes. These methods work with the mammalian egg and cells in culture, but the reproductive process in the chicken severely limits the use of these techniques in the ovum or newly fertilized ovum of the chicken, b) A delivery system currently being explored for gene transfer in the chicken is the use of avian retrovirus as a "piggy-back" carrier. The life style of the retrovirus is to infect the cell and make a cDNA copy of its genome that inserts into the chicken chromosome as a provirus gene. A reconstructed virus, with all its insertion properties intact and its infectious attributes deleted, would be an ideal gene-shuttle-vector, as it would make its own way into the nucleus of the cell with considerable certainty of insertion into the chromosome. A major portion of this symposium is given over to retrovirus technology that is well covered by Crittenden (1986), Hughes (1986), Salter (1986), and Shuman (1986). One cannot help being impressed by the imaginative research in this area. Little more will be said here, except to point out that retroviruses are successful vectors for transferring genes into mice and appear extremely promising in the chicken.
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TK, and antichicken T K inhibits chicken TK p r o d u c t i o n b u t n o t HSV-TK. If this technology is developed t o an operational p o i n t in p o u l t r y , o n e can envision application t o suppress expression of several undesirable traits such as broodiness in t u r k e y s , fat deposition in chicken broilers, a n d cholesterol in eggs.
genetics, this stage of t h e game is one of t h e m o r e exciting times of all. T h e investment in molecular biology is certain t o pay off in genetically improved stock in one w a y or another.
SECURING FUNDAMENTAL KNOWLEDGE ABOUT GENE REGULATION
Crittenden, L. B., 1986. Identification and cloning of genes for insertion. Poultry Sci. 65:1468-1473. Hughes, H., 1986. Vectors for gene transfer. Poultry Sci. 65: (in press). Isant, J. G., and H. Weintraub, 1985. Constitutive and conditional suppression of exogenous and endogenous genes by antisense RNA. Science 220: 345-352. Jones, D. D., 1985. Commercialization of gene transfer in food organisms: A science-based regulatory model. Food Drug Cosmet. Law J. 40:477—493. Salter, D. W., 1986. Gene insertion by recombinant avian retrovirus and retroviral DNA. Poultry Sci. 65:(in press). Shuman, R. M., 1982. Transfer of primoridal germ cells (PGC's) in the chicken. M. S. Thesis. Univ. of Minnesota. Shuman, R. M., and R. M. Shoffner, 1985. Gene transfer by avian retroviruses. Poultry Sci. 65: 1437-1444. Soller, M., and J. S. Beckman, 1986. Restriction fragment length polymorphisms in poultry breeding. Poultry Sci. 65:1474-1488.
REFERENCES
CONCLUSIONS When viewing t h e w h o l e of b i o t e c h n o l o g y research in animals, p o u l t r y is lagging behind. This is partly because there is little critical reason for d e v e l o p m e n t of such technology, as e m b r y o transfer or cloning, since egg transp o r t capability, relatively s h o r t generation turnover, highly inbred lines, and genetic information from family structure satisfy t h e benefits gained from these procedures in t h e large m a m m a l i a n species. Cryopreservation of gametes is currently only possible with spermat o z o a of birds, a n d while this process is i m p o r t a n t from a germ plasma preservation s t a n d p o i n t , it has n o t been a critical factor for t h e industry. Access t o t o t i p o t e n t cells in t h e avian r e p r o d u c t i o n cycle is a major p r o b l e m c o m p a r e d t o t h e ability t o m a n i p u l a t e and microinject t h e m a m m a l i a n egg. Once a satisfactory delivery system is developed, one may expect considerable productivity in b o t h f u n d a m e n t a l and application research. Even after gene transfer is accomplished, several problems remain t o be solved. T h e n u m b e r of gene copies and sites of insertion are at present uncontrollable. F u n c t i o n a l i t y is problematical, and knowledge a b o u t gene regulation is obscure. Insertion of a p p r o p r i a t e p r o m o t e r s upstream t o t h e transferred sequence and o t h e r considerations still have t o be w o r k e d o u t before t h e perfect functional gene is secured. F o r one w h o has had t h e o p p o r t u n i t y to observe and experience some of t h e contributions of classical genetics, quantitative genetics, cytogenetics, and a small a m o u n t of molecular
ADDITIONAL REFERENCES Overviews Beltsville Symposium X Abstracts, 1985. Biotechnology for solving agricultural problems. Beltsville, MD. May 5 - 9 . Frankham, R., and M. R. Gillings, 1984. Molecular biology and its application to domestic animals. Animal genetic resources: cryogenic storage of germplasm and molecular engineering. FAO Animal Production and Health Paper 44/2. Freeman, B. M., and L. I. Messer, 1985. Genetic manipulation of the domestic fowl — A review. World's Poult. Sci. J. 41:124-132. Genetic engineering of animals: An agricultural perspective, 1985. Univ. of California, Davis. Sept. 9 - 1 2 . Purchase, H. G., 1985. Future applications of biotechnology in poultry. Proc. Annu. Mtg. Am. Vet. Med. Assoc, Las Vegas, NE. July 23. Shuman, R., and R. N. Shoffner, 1982. Potential genetic modification in the chicken, Gallus domesticus. In Proc. 2nd World Congr. Genetics Applied to Livestock Production VI. 157—163. Wilson, T., 1984. Expression vectors: More protein from mammalian cells. Bio/Tech. 2:753—755. Microbial Manipulation-Vaccine
Production
Brown, F., 1985. Peptides as the next generation of foot-and-mouth disease vaccines. Bio/Tech. 3: 445-448. Kaper, J. B., H. Lockman, M. M. Baldini, and M. M.
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Gene expression in t h e specialized cells of t h e chicken m u s t have t h o u s a n d s of genes t h a t are t u r n e d o n and off in a regulated way. Very little is u n d e r s t o o d about transcriptional regulation in animal cells so t h a t transferred genes w o u l d provide an o p p o r t u n i t y t o learn m o r e a b o u t t h e complicated regulation process of t h e chicken.
SYMPOSIUM: MOLECULAR APPROACHES TO POULTRY BREEDING
Recombinant DNA
Technology
Maniatis, T., E. F. Fritsch, and J. Sambrook, 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Watson, J. D., J. Tooze, and D. T. Kurtz, 1983. Recombinant DNA: A Short Course. Scientific American Books, W. H. Freeman and Co., NY. Location of Genes and Restriction Fragment Length Polymorphism (RFLP) Harper, M. E., and G. F. Saunders, 1981. Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization. Chromosoma 83:431—439. Isobe, M., J. Erickson, B. S. Emanuel, P. C. Nowell, and C. M. Croce, 1985. Location of gene for (5 subunit of human T-cell receptor at band 7q35, a region prone to rearrangements in T cells. Science 228:580-582. Skolnick, M. H., and R. White, 1982. Strategies for detecting and characterizing restriction fragment length polymorphisms (RFLP's). Cytogenet. Cell Genet. 32:58-67. Soller, M., and J. S. Beckman, 1982. Restriction fragment length polymorphisms and genetic improvement. Pages 396—404 in Proc. 2nd World Congr. Genetics Applied to Livestock Production VI. Vogelstein, B., E. R. Fearon, S. R. Hamilton, and A. P. Feinberg, 1985. Use of restriction fragment length polymorphisms to determine the clonal origin of human tumors. Science 227:642—645. Young, R. A., and R. W. Davis, 1983. Efficient isolation of genes using antibody probes. Proc. Natl. Acad. Sci. USA. 80:1194-1198. Biohazards Brill, W. J., 1985. Safety concerns and genetic engineering in agriculture. Science 229:381—384. Markle, G. E., and S. S. Robin, 1985. Biotechnology and the social reconstruction of molecular biology. Bioscience 35:220-225.
Gene Therapy Tangley, L., 1985. Gearing up for gene therapy. Bioscience 35:8—10. Antisense
RNA
Izant, J. G., and H. Weintraub, 1984. Inhibition of thymidine kinase genes expression by anti-sense RNA: A molecular approach to genetic analysis. Cell 36: 1007-1015. Klausner, A., 1985. Turning off unwanted genes with anti-RNA. Bio/Technology, 3:763-764. Retrovirus as Vectors and Associated
Technology
Hughes, S., and E. Kosik, 1984. Mutagenesis of the region between env and src of the SR-A strain of Rous sarcoma virus for the purpose of constructing helper-independent vectors. Virology 136:89-99. Jaenisch, R. J., D. Janher, P. Nobis, I. Simon, J. Lohler, K. Harbers, and D. Grotkopp, 1981. Chromosomal position and activation of retroviral genomes inserted into the germ line of mice. Cell 24:519-529. Miller, A. D., R. J. Eckner, D. J. Jolly, T. Friedman, and I. M. Verma, 1984. Expression of a retrovirus encoding human HPRT in mice. Science 225: 630-632. O'Rear, J. J., S. Mizutani, G. Hoffman, M. Fiandt, and H. M. Temin, 1980. Infectious and non-infectious clones of the provirus of SNV differ in cellular DNA and are apparently the same in viral DNA. Cell 20:423-430. Shuman, R. M., 1984. The avian retrovirus as potential vector for gene transfer in the chicken. Ph.D. Thesis. Univ. of Minnesota. Gene Transfer Experiments Frahley, R., S. Subramani, P. Berg, and D. Papahadjopoulos, 1980. Introduction of liposomeencapsulated SV-40 DNA into cells. J. Biol. Chem. 225:10431-10435. Gill, J. A., J. P. Sumpter, E. M. Donaldson, H. M. Dye, L. Souza, T. Berg, J. Wypych, and K. Langeley, 1985. Recombinant chicken and bovine growth hormones accelerate growth in aquacultured juvenile pacific salmon Oncorhynchus kisutch. Bio/Tech. 3:643-646. Gordon, J. W., and F. H. Ruddle, 1981. Integration and stable germline transmission of genes injected into mouse pronuclei. Science 214:1244—1246. Gordon, J. W., G. A. Scangos, D. J. Plotkin, J. A. Barbosa, and F. H. Ruddle, 1980. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci., USA 77:7380-7384. Klebe, R. J., J. V. Harriss, D. P. Hanson, and C. J. Gauntt, 1984. High-efficiency polyethelene glycol-mediated transformation of mammalian cells. Somat. Cell Mol. Genet. 10:495-502. Krowtiris, T. G., and G. M. Cooper, 1981. Transforming activity of human tumor DNAs. Proc. Natl. Acad. Sci. USA 78:1181-1184. McKnight, G. S., R. E. Hammer, E. A. Kuenzel, and R. L. Brinster, 1983. Expression of the chicken
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Levine, 1*984. A recombinant live oral cholera vaccine. Bio/Tech. 2:345-349. Kleid, D. G., D. Yansura, B. Small, D. Dowbenko, D. M. Moore, M. J. Grubman, P. D. McKercher, D. O. Morgan, B. H. Robertson, and H. L. Bachrach, 1985. Cloned viral protein vaccine for footand-mouth disease: Responses in cattle and swine. Science 214:1125-1129. Laskey, L. A., D. Dowbenko, C. C. Simonsen, and P. W. Berman, 1984. Protection of mice from lethal herpes simplex virus infection by vaccination with a secreted form of cloned glycoprotein D. Bio/Tech. 2:527-532. Mackett, M., T. Yilma, J. K. Rose, and B. Moss, 1985. Vaccinia virus recombinants: expression of VSV genes and protective immunization of mice and cattle. Science 227:433-435. Roizman, B., and F. J. Jenkins, 1985. Genetic engineering of novel genomes of large DNA viruses. Science 2 2 9 : 1 2 0 8 - 1214.
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transferrin gene in transgenic mice. Cell 34: 335-341. Palmiter, R. D., R. L. Brinster, R. E. Hammer, M. E. Trumbauer, M. G. Rosenfeld, N. C. Birnberg, and R. M. Evans, 1982. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 300:611-615. Wagner, T. E., P. C. Hoppe, J. D. Jollick, D. R. SchoU, R. L. Hodinka, and J. B. Gault, 1981. Microinjection of a rabbit B-globin gene into zygotes and its subsequent expression in adult mice and
their offspring. 78:6376-6380.
Proc. Natl. Acad. Sci. USA
Growth Factors and Oncogenes Kamata, T., and J. R. Feramisco, 1984. Epidermal growth factor stimulates guanine nucleotide binding activity and phosphorylation of ras oncogene proteins. Nature 310:147—150. Kris, R. M., T. A. Libermann, A. Avivi, and J. Schlessinger, 1985. Growth factors, growth-factor receptors and oncogenes. Bio/Tech. 3:135—140.
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1986 Poultry Science 65:1489-1496 INTRODUCTION Molecular genetic t e c h n o l o g y is n o w an integral p a r t of t h e inquiry into biological functions involved in inheritance, cytogenetics, i m m u n o l o g y , endocrinology, nutrition, behavior, r e p r o d u c t i o n , and animal health. C o m m e r c i a l applications already include t h e p r o d u c t i o n of pharmaceuticals, f e r m e n t a t i o n products, and vaccines. A considerable p o r t i o n of current f u n d a m e n t a l biological research reduces t o genetics in t h e sense t h a t d e o x y ribonucleic acid (DNA) and ribonucleic acid ( R N A ) nucleotide sequences have b e e n identified for a wide variety of biological c o m p o u n d s including globins, chick a l b u m e n , platelet growth factor, specific i m m u n o p e p t i d e s , g r o w t h h o r m o n e , insulin, interferon, and o t h e r comp o u n d s . Even t h e c o m p l e t e g e n o m e s of s o m e microorganisms have b e e n characterized. Currently, t h e enthusiasm for genetic engineering in p o u l t r y is t h e p o t e n t i a l to use gene transfer t e c h n o l o g y for genetic improve-
m e n t . However, we m u s t recognize t h a t molecular genetics is n o t a panacea or a r e p l a c e m e n t of conventional p o u l t r y breeding t e c h n o l o g y . It is a marvelous t o o l for uncovering new knowledge a n d adds t o our ability t o advance genetic i m p r o v e m e n t . Molecular genetic t e c h n o l o g y involves t h e m a n i p u l a t i o n of genes in vitro w i t h r e c o m b i n a n t D N A m e t h o d o l o g y . T h e gene r e p l a c e m e n t p r o c e d u r e substitutes a gene sequence of cloned r D N A i n t o c h r o m o s o m a l D N A of a cell or organism, either b y first removing c h r o m o s o m a l D N A and replacing it b y r e c o m b i n a n t D N A (rDNA), or merely adding in t h e extra rDNA t o create novel g e n o t y p e s . Mutagenesis is a n o t h e r manipulative m e t h o d w h e r e b y t h e gene sequence is altered in vitro, t h e n followed b y r e i n t r o d u c t i o n into t h e cell or organism — in effect, t h e creation and insertion of a new gene. A t h i r d system for altering g e n o t y p e s is t h e fusion gene t e c h n i q u e accomplished b y fusing two gene sequences or p o r t i o n s of t w o genes into a single gene t h a t has altered transcription capability.
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Paper No. 14729 in the Scientific Journal Series of the Minnesota Agricultural Experiment Station. 1489
BENEFITS OTHER THAN DIRECT GENE TRANSFER A n i m p o r t a n t area of genetic engineering
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ABSTRACT Genetic engineering of microrganisms to produce more efficient vaccines, immunizing polypeptides, monoclonal antibodies, microbes with enhanced biodegradable capabilities, and pharmaceuticals have potential usefulness for poultry producers and researchers. Molecular technology can be used for chromosome mapping. Application of gene transfer technology to poultry requires an integrated program, including molecular genetics expertise, cell biology-embryology knowledge, and breeding capabilities. A successful gene transfer in poultry promises an increase in genetic variability. A gene theoretically can be inserted directly into selected stocks without extraneous genetic material. There is the exciting potential for inserting genes from other species. The recombinant deoxyribonucleic acid (DNA) technology for isolating and cloning a gene sequence can be readily applied to poultry. Cloned DNA for a desired gene sequence requires a delivery system to get the sequence into the germ line. A retrovirus vector that includes the sequence appears to be the system of choice for oviparous species at this time. Germ line target cells are primordial germ cells, spermatozoa, ova, and early embryonic cells that will give rise to gonads that have a chromosome with the inserted sequence. Evaluation of gene transfers at the molecular level will determine if insertion is accomplished, as well as the number and location. A major portion of a molecular genetics program will include breeding evaluation for expression, stability, and usefulness, including all the conventional procedures for multiplication, selection and structuring of populations for crossing, and field testing. (Key words.- poultry, molecular genetics, recombinant deoxyribonucleic acid, delivery vectors, antisense ribonucleic acid, breeding application)
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On the other hand, the benefits for research application have considerable promise. Gene cloning is part of basic inquiry into physiological function and is becoming almost routine, and, when applied to poultry, will greatly increase our understanding of the fundamental biology of birds. Currently, securing pituitary hormones is not only a tedious process, but only minute amounts are obtained from processing immense numbers of chicken or turkey pituitaries. Cloning the gonad-stimulating hormone (GSH) gene, for example, inserting it into bacteria or cells in culture would make it possible to produce gram quantities of this or similar products. GENE MAPPING Chromosome Location. The chicken gene map has about 12 linkage groups and only a few are assigned to specific chromosomes. The major portion of the genes in the current linkage groups are those for morphological traits. Assignment to chromosomes by conventional cytogenetic methods, somatic cell hybrids, or rearrangement marker chromosome methods has not been very fruitful
because of high recombination frequency. Molecular technology utilizing radioactive marker probes, avidin-biotin probes, and similar methods now make it possible to identify single copy DNA on in situ treated chromosome preparations or to use restriction fragment length polymorphisms (RFLP) in gene-mapping studies. The gene map for chickens and other domestic poultry is expected to expand rapidly with fairly precise chromosomal locations. At the outset, the gene map will consist primarily of traits recognizable in electrophoretic gels and similar techniques, and, in time, these traits will be linked to morphological linkage groups and possibly major genes or blocks of genes influencing quantitative traits. Marker-Assisted Selection. Single genes may be only a few kilobases (kb) in length, but flanking RFLP sequences could multiply the recognizable region several times, thus increasing the number of labeled hybridizing probes as well as the receptive recognition sites on the chromosome. Morris Soller (Soller and Beckman, 1986) proposes to take advantage of the RFLP technology to identify genes or regions on the chromosome for marker-assisted selection of quantitative traits. Sex Probe. Vent sexing, plumage colors, and feather sexing at hatching is routine, because sexual dimorphism of domestic poultry makes it relatively easy to distinguish sex. Sexual dimorphism at all ages is absent or vague species, zoological specimens, and pets. The W chromosome of the heterogametic females can be recognized in karyotype preparations especially if assisted by the cytochemical differentiation C-banding technique. Determination of sex in very early embryos prior to gonadal differentiation can also be accomplished by cytochemical differentiation methods, if chromosomal preparations are available. Even though the cytogenetic techniques are workable, the identification of a W chromosome sequence that can be recognized with a specific probe would be exceedingly convenient for determining sex of cells from all types of specimens.
SOMATIC CELL TRANSPLANTS This system is designed for gene therapy, whereby genetically defective cells are made "normal" by insertion of the nondefective alleles into cells in culture and then transplanted back into the organ where the defective
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that can benefit the poultry industry involves the restructuring of microorganisms to create novel genomes with improved immunization capability. Several strategies have been used, or proposed, to modify viral genomes for vaccines or as vectors for genes that specify antigens for prevention of infectious diseases. A vaccinia virus, carrying a foreign gene sequence with specific immunizing properties, could conceivably be a more efficient vaccine for those diseases of poultry where only killed organisms are now safe to use. The identification of specific polypeptide sequences with immunizing capacity against bacteria and protozoa may lead to products protecting poultry from infection by these organisms. Reconstruction of microbes with enhanced biodegradable capacities, such as those degrading oil and halogenated compounds, may be useful in poultry waste lagoons and methane gas production systems. Pharmaceutical products such as monoclonal antibody for poultry health may have limited application, because the small individual worth may limit the use of costly therapeutic application. The value of injectable compounds such as growth hormone will depend upon the trade-off between costs of compound and injection vs. gains in performance.
SYMPOSIUM: MOLECULAR APPROACHES TO POULTRY BREEDING
GENE TRANSFER IN POULTRY To take advantage of the opportunities of molecular biology in poultry research, and especially gene transfer technology, one has to bring together an integrated program involving the expertise of molecularly trained personnel, cell biology-embryology knowhow, and, finally, but not least, breeding and genetics for evaluation and propagation. This can be accomplished in one or several laboratories, but the essential ingredients have to come together for total application. Potential Advantages. 1. Genetic variability would be increased and so would the possibility of an advantageous genetic change. 2. A gene could be inserted directly into selected, wellcharacterized stocks such as grandparent lines, inbred lines, and purebred stocks. The tedious and time-consuming efforts of many backcrosses required in the conventional gene transfer would be avoided. 3. The possibility of overcoming fertility barriers and inserting exotic genes from another species has considerable interest. 4. Unfavorable linkage situations could be broken through rearrangement and reinsertion. 5. The chromosome map would be increased as it is important to locate the original gene locus as well as that of an "add in" gene. Disadvantages. Molecular gene transfer application research is a high-cost, high-risk venture. Few poultry research units have the facilities, equipment, or personnel necessary to pursue molecular technology, and it requires considerable investment just to begin a program. There is no guarantee that gene transfer will result in dramatic genetic improvement of poultry. The frequency of failures may far
exceed successes. There is no doubt that structural genes will be inserted into the genome, although there are several technical problems to be overcome before this process becomes routine. At this point in time, identification of useful genes has to be accomplished, as well as learning about gene regulation in the complicated feedback systems of the chicken. However, I consider these to be difficult technical problems that will be solved in time, rather than complete barriers to useful gene transfers. Possible Biohazards. The probability is exceedingly low that a transformed chicken will endanger either other organisms or the environment, because they are highly contained and can be readily destroyed if necessary. Modern highly selected strains of domestic chickens, turkeys, ducks, and geese have lost a portion of their genetic adaptability to survive in the "wild." Furthermore, the inserted gene may make them even less adaptive for survival without man's husbandry. Perhaps the greatest danger could be loss of germplasm. Suppose, for example, that a very favorable genetic transfer is made into an egg production stock, and all other stocks were discarded. Subsequently, it was found the transformed birds were extremely susceptible to a particular infection, and alternative germ plasm is unobtainable. This possibility seems exceedingly remote, because it is a well-recognized problem and will be guarded against. Release of genetically engineered organisms into the environment is a concern, and precautionary guidelines have been instituted and rigorously followed to control possible problems with engineered laboratory strains of microorganisms. Restructured RNA and DNA viruses, as vectors capable of delivering genes and at the same time being capable of selfperpetuation and spread, will require careful monitoring. However, vectors have been, and future ones can be, restructured through genetic engineering, so that they are incapable of reproduction or causing infection. Jones (1985), discusses some possible situations that will require evaluation for product safety in transfected food animals. When the host's genome is altered by a gene insert that acts independently of the recipients genome, it gives an exogenous expression that may be desirable or undesirable depending upon its end product. A viral vector that retains ability to produce infectious virus, or an insert that gives a car-
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cells have been killed or inactivated in order that the transplant cells can grow and proliferate. The practical advantages of this system is to get around the usual immunological complications of transplants or into regions where organs cannot be removed and replaced. Some success has been achieved with bone marrow cells in leukemic mice, and there is considerable interest for application in human medicine. Because of the limited value of the individual chicken, even if this technology is perfected, it would probably be of little value to the poultry industry. A more feasible alternative would be a gene transfer to prevent infection, such as a resistance gene that acts endogenously to insure healthy birds.
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cinogenic product, would be extremely undesirable. Alternatively, the exogenous gene could produce a desirable effect by improving the animal's well-being. If the replicated inserts come under the control of the host's genome, the expression product is considered as endogenous. The resulting expression may be either favorable or unfavorable depending upon the nature of the product coded for by the insert.
Recombinant Deoxyribonucleic Acid Technology. Step Number one is to decide on a gene with potential value such as growth hormone or similar compound. The preference would be one with a short uncomplicated sequence, as it would be easier to identify in a genomic library, and insertion into a plasmid vector would be less complicated with a greater probability for chromosome insertion. The next maneuver is to identify and isolate the gene sequence of choice. The method of least trouble is to beg one from another laboratory. If a homologous, or nearly homologous, sequence is available from another species, for example, bovine growth hormone, the isolation of the chicken growth hormone is simplified. If these alternatives are not available, one has to start from scratch, use restriction enzymes to cleave the DNA, form a genomic library, and go through the tedious search for the desired sequence using probes designed to be complementary to a known protein sequence, or through antibody probing of an expression library. The next step is to isolate the desired sequence through the applications of recombinant DNA (rDNA) technology for multiplication of gene copies. Sequences are recombined into the chromosome of a plasmid such as pBR322, a phage, or a cosmid. Cosmids are plasmid-phage complexes usually able to hold a larger sequence than plasmids. Both plasmids and phage live and multiply in bacteria, and the laboratory strains of E. coli are usually the organisms of choice for amplification. Clones of bacteria, each with a different insertion, are grown and tested for the presence of the plasmids with the desired sequence. Once isolated, the plasmids with the gene are amplified in E. coli cultures for producing many gene copies.
Target Cells. Totipotent cells are those whose chromosomal complement will eventually be in some or all subsequent cells of the organism. The target cells of importance are the gonadal cells, a) The primordial germ cells (PGC) that arise early in embryonic development as receptors of transferred genes would be expected to be progenitors of cells producing gametes carrying the transferred gene. A gonad with transplanted PGC, carrying gene sequence inserts, theoretically would produce gametes with the transferred gene. Shuman (1983) developed a successful transplant technique, but donor chromosomally marked gametes were not identified in subsequent test matings. Consequently, while this route is an appealing one for gene transfer and other manipulations, several technical problems will have to be mastered before this system becomes useful, b) Gametes, either sperm or egg, with an inserted gene sequence, would contribute that segment to all subsequent cells in the zygote. For sperm to be efficient vectors, some method of mass treatment would have to be devised. Some thought and effort has been given to developing a viral vector (retrovirus construct for example) that would attach to sperm, thereby be transported into the egg, and thus have an opportunity for the vector to be incorporated into a chromosome of the subsequent zygote. The avian ovum is located on an extremely large yolk, and, upon ovulation, the ovum is picked up almost immediately by the infundibulum where fertilization occurs shortly before the ovum begins its journey down the oviduct to complete egg formation. Consequently, manipulation of the ovum is difficult for injection or similar procedures, c) Incorporation of calcium phosphate precipitated rDNA into the cell nucleus results in transfection of donor rDNA into the chromosomal DNA of the host cell. The newly laid fertile egg has an embryo that has developed to the blastocoele stage. At this stage or shortly thereafter, the embryo is presumed to contain inner cell mass (ICM) cells that are totipotent or at least pluripotent so that rDNA transfection of these cells may be expected to have subsequent gonadal cells carrying the transferred gene. Because embryos of this stage are readily available, there is considerable attraction for exploring this route for rDNA transfection.
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OPERATIONAL STEPS IN GENE TRANSFER APPLIED TO POULTRY BREEDING
DEVELOPING A DELIVERY SYSTEM FOR INSERTION INTO THE CHICKEN GENOME
SYMPOSIUM: MOLECULAR APPROACHES TO POULTRY BREEDING
EVALUATION FOR EXPRESSION AND FUNCTIONALITY Determination of Insertion by Molecular Technology. Following gene transfer attempts, one wishes to know if the transfer was accomplished. A successful insert into a chromosome is expected to be transmitted to progeny. The DNA secured from tissue or nuclei of red blood cells of chicks can be probed by several means utilizing dot-blot electrophoresis, autoradiographic labeled DNA (same sequence as inserted), antibiotin antibody fluoresence, or other methods to determine if the intended inserted sequence is present in the chromosomal DNA. There are reasons for making the DNA determinations besides that of merely detecting presence of the insert. The number of inserted copies may vary in number as well as chromosomal location, which, at this point, is assumed to be random. None, one, or several inserts may be expressed. Location of inserts by hybridization probes to the chromosomal regions, especially in relation to the indigenous genes, is important. Some interesting ratios
could occur if an inserted gene expresses as well as the indigenous locus. In addition to segregation ratios in expression, there would also be an independent assortment expression situation if the insert is on a different chromosome or on the same chromosome in a different locus. Dosage effects may also be involved in expression. Poultry Breeding Technology. Let us suppose that a desirable gene has been transferred and is expressed in one of the progeny. To start, we have one transfected individual that is heterozygous as insertion is expected in only one of the pair of homologus chromosomes. Dominant expression would be recognized immediately, while recessiveness requires further matings for expression. From now on in the application process, we have to depend upon sexual propagation to multiply the newly created genome with its heterozygous insert. Only 50% of the progeny will carry the new gene so all the appropriate matings, recording, and selection of parents will follow conventional breeding procedures. In successive generations, we will need to observe for stability. Does the new gene just fade away? Does it act like a transposon (jumping gene), moving from chromosome to chromosome and changing expression as it changes location? What will be the effect on other traits? Will the energy required for increased expression detract from other traits? Is the inserted gene an additional load on an already established genome? Other types of interaction effects are possible. If the new gene passes all the desirability tests, it has to be incorporated into a selected stock. Hopefully, the insert will be made into a select stock so that the transferred gene is already where it is wanted. Test crosses, field tests, and all the usual breeding work necessary for evaluating a commercial product will be required. It will take some time before the "wonder" gene appears in millions of chickens. RECOMBINANT RIBONUCLEIC ACID The technology to recombine RNA fragments has lead to what is called ANTISfiNSE RNA. The RNA is made double stranded in opposing directions and suppresses translation of RNA by forming RNA/RNA hybrids. The activity appears to be specific, as Izant and Weintraub (1985) found that antisense Herpes Simplex virus-thymidine kinase (HSV-TK) inhibits HSV-TK production but not chicken
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Delivery Systems, a) Common methods of transfecting rDNA into the nucleus are microinjection, calcium phosphate precipitate with enhancement, and liposomes. These methods work with the mammalian egg and cells in culture, but the reproductive process in the chicken severely limits the use of these techniques in the ovum or newly fertilized ovum of the chicken, b) A delivery system currently being explored for gene transfer in the chicken is the use of avian retrovirus as a "piggy-back" carrier. The life style of the retrovirus is to infect the cell and make a cDNA copy of its genome that inserts into the chicken chromosome as a provirus gene. A reconstructed virus, with all its insertion properties intact and its infectious attributes deleted, would be an ideal gene-shuttle-vector, as it would make its own way into the nucleus of the cell with considerable certainty of insertion into the chromosome. A major portion of this symposium is given over to retrovirus technology that is well covered by Crittenden (1986), Hughes (1986), Salter (1986), and Shuman (1986). One cannot help being impressed by the imaginative research in this area. Little more will be said here, except to point out that retroviruses are successful vectors for transferring genes into mice and appear extremely promising in the chicken.
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TK, and antichicken T K inhibits chicken TK p r o d u c t i o n b u t n o t HSV-TK. If this technology is developed t o an operational p o i n t in p o u l t r y , o n e can envision application t o suppress expression of several undesirable traits such as broodiness in t u r k e y s , fat deposition in chicken broilers, a n d cholesterol in eggs.
genetics, this stage of t h e game is one of t h e m o r e exciting times of all. T h e investment in molecular biology is certain t o pay off in genetically improved stock in one w a y or another.
SECURING FUNDAMENTAL KNOWLEDGE ABOUT GENE REGULATION
Crittenden, L. B., 1986. Identification and cloning of genes for insertion. Poultry Sci. 65:1468-1473. Hughes, H., 1986. Vectors for gene transfer. Poultry Sci. 65: (in press). Isant, J. G., and H. Weintraub, 1985. Constitutive and conditional suppression of exogenous and endogenous genes by antisense RNA. Science 220: 345-352. Jones, D. D., 1985. Commercialization of gene transfer in food organisms: A science-based regulatory model. Food Drug Cosmet. Law J. 40:477—493. Salter, D. W., 1986. Gene insertion by recombinant avian retrovirus and retroviral DNA. Poultry Sci. 65:(in press). Shuman, R. M., 1982. Transfer of primoridal germ cells (PGC's) in the chicken. M. S. Thesis. Univ. of Minnesota. Shuman, R. M., and R. M. Shoffner, 1985. Gene transfer by avian retroviruses. Poultry Sci. 65: 1437-1444. Soller, M., and J. S. Beckman, 1986. Restriction fragment length polymorphisms in poultry breeding. Poultry Sci. 65:1474-1488.
REFERENCES
CONCLUSIONS When viewing t h e w h o l e of b i o t e c h n o l o g y research in animals, p o u l t r y is lagging behind. This is partly because there is little critical reason for d e v e l o p m e n t of such technology, as e m b r y o transfer or cloning, since egg transp o r t capability, relatively s h o r t generation turnover, highly inbred lines, and genetic information from family structure satisfy t h e benefits gained from these procedures in t h e large m a m m a l i a n species. Cryopreservation of gametes is currently only possible with spermat o z o a of birds, a n d while this process is i m p o r t a n t from a germ plasma preservation s t a n d p o i n t , it has n o t been a critical factor for t h e industry. Access t o t o t i p o t e n t cells in t h e avian r e p r o d u c t i o n cycle is a major p r o b l e m c o m p a r e d t o t h e ability t o m a n i p u l a t e and microinject t h e m a m m a l i a n egg. Once a satisfactory delivery system is developed, one may expect considerable productivity in b o t h f u n d a m e n t a l and application research. Even after gene transfer is accomplished, several problems remain t o be solved. T h e n u m b e r of gene copies and sites of insertion are at present uncontrollable. F u n c t i o n a l i t y is problematical, and knowledge a b o u t gene regulation is obscure. Insertion of a p p r o p r i a t e p r o m o t e r s upstream t o t h e transferred sequence and o t h e r considerations still have t o be w o r k e d o u t before t h e perfect functional gene is secured. F o r one w h o has had t h e o p p o r t u n i t y to observe and experience some of t h e contributions of classical genetics, quantitative genetics, cytogenetics, and a small a m o u n t of molecular
ADDITIONAL REFERENCES Overviews Beltsville Symposium X Abstracts, 1985. Biotechnology for solving agricultural problems. Beltsville, MD. May 5 - 9 . Frankham, R., and M. R. Gillings, 1984. Molecular biology and its application to domestic animals. Animal genetic resources: cryogenic storage of germplasm and molecular engineering. FAO Animal Production and Health Paper 44/2. Freeman, B. M., and L. I. Messer, 1985. Genetic manipulation of the domestic fowl — A review. World's Poult. Sci. J. 41:124-132. Genetic engineering of animals: An agricultural perspective, 1985. Univ. of California, Davis. Sept. 9 - 1 2 . Purchase, H. G., 1985. Future applications of biotechnology in poultry. Proc. Annu. Mtg. Am. Vet. Med. Assoc, Las Vegas, NE. July 23. Shuman, R., and R. N. Shoffner, 1982. Potential genetic modification in the chicken, Gallus domesticus. In Proc. 2nd World Congr. Genetics Applied to Livestock Production VI. 157—163. Wilson, T., 1984. Expression vectors: More protein from mammalian cells. Bio/Tech. 2:753—755. Microbial Manipulation-Vaccine
Production
Brown, F., 1985. Peptides as the next generation of foot-and-mouth disease vaccines. Bio/Tech. 3: 445-448. Kaper, J. B., H. Lockman, M. M. Baldini, and M. M.
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Gene expression in t h e specialized cells of t h e chicken m u s t have t h o u s a n d s of genes t h a t are t u r n e d o n and off in a regulated way. Very little is u n d e r s t o o d about transcriptional regulation in animal cells so t h a t transferred genes w o u l d provide an o p p o r t u n i t y t o learn m o r e a b o u t t h e complicated regulation process of t h e chicken.
SYMPOSIUM: MOLECULAR APPROACHES TO POULTRY BREEDING
Recombinant DNA
Technology
Maniatis, T., E. F. Fritsch, and J. Sambrook, 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Watson, J. D., J. Tooze, and D. T. Kurtz, 1983. Recombinant DNA: A Short Course. Scientific American Books, W. H. Freeman and Co., NY. Location of Genes and Restriction Fragment Length Polymorphism (RFLP) Harper, M. E., and G. F. Saunders, 1981. Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization. Chromosoma 83:431—439. Isobe, M., J. Erickson, B. S. Emanuel, P. C. Nowell, and C. M. Croce, 1985. Location of gene for (5 subunit of human T-cell receptor at band 7q35, a region prone to rearrangements in T cells. Science 228:580-582. Skolnick, M. H., and R. White, 1982. Strategies for detecting and characterizing restriction fragment length polymorphisms (RFLP's). Cytogenet. Cell Genet. 32:58-67. Soller, M., and J. S. Beckman, 1982. Restriction fragment length polymorphisms and genetic improvement. Pages 396—404 in Proc. 2nd World Congr. Genetics Applied to Livestock Production VI. Vogelstein, B., E. R. Fearon, S. R. Hamilton, and A. P. Feinberg, 1985. Use of restriction fragment length polymorphisms to determine the clonal origin of human tumors. Science 227:642—645. Young, R. A., and R. W. Davis, 1983. Efficient isolation of genes using antibody probes. Proc. Natl. Acad. Sci. USA. 80:1194-1198. Biohazards Brill, W. J., 1985. Safety concerns and genetic engineering in agriculture. Science 229:381—384. Markle, G. E., and S. S. Robin, 1985. Biotechnology and the social reconstruction of molecular biology. Bioscience 35:220-225.
Gene Therapy Tangley, L., 1985. Gearing up for gene therapy. Bioscience 35:8—10. Antisense
RNA
Izant, J. G., and H. Weintraub, 1984. Inhibition of thymidine kinase genes expression by anti-sense RNA: A molecular approach to genetic analysis. Cell 36: 1007-1015. Klausner, A., 1985. Turning off unwanted genes with anti-RNA. Bio/Technology, 3:763-764. Retrovirus as Vectors and Associated
Technology
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