I. Funding for the program is essentially doubled. Approximately $3 million is expected to be available to support research grants from the BRARGP in fiscal year 2003.

II. Research Priorities for the BRARGP have been expanded to include:

• (1) Research designed to identify and develop appropriate management practices to minimize physical and biological risks associated with genetically engineered animals, plants, and microorganisms.

• (2) Research designed to develop methods to monitor the dispersal of genetically engineered animals, plants, and microorganisms.

• (3) Research designed to further existing knowledge with respect to the characteristics, rates, and methods of gene transfer that may occur between genetically engineered animals, plants, and microorganisms and related wild and agricultural organisms.

• (4) Environmental assessment research designed to provide analysis, which compares the relative impacts of animals, plants, and microorganisms modified through genetic engineering to other types of production systems.

• (5) Other areas of research designed to further the purposes of this program.

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REPORT ON TALK TO THE UNITED NATIONS
Jennifer Thomson

On November 6, 2002, I participated in the second lecture in a series arranged at the request of Kofi Annan, Secretary-General of the United Nations. The title of my lecture was Genetically Modified Crops for Developing Countries. There were two speakers, Dr Daphne Preuss of the University of Chicago and myself. The audience consisted of UN delegates, UN staff members, and various NGOs.

Before the talks, the Secretary-General hosted a lunch, which was also attended by the Deputy Secretary-General, Madame Louise Frechette, the Assistant Secretary-General, Michael Doyle, and the President of the US National Academy of Science, Dr. Bruce Alberts. This luncheon provided an opportunity for us to discuss some of the issues that would be raised during the lectures with a few of the UN delegates who are particularly interested in this topic.

Dr. Preuss, who works on whole chromosomes in plants, gave an overview of classical plant breeding, explaining how genetic modification differed and describing the relative advantages of each breeding method. She also discussed intellectual property and market forces. I focused on how GM crops can make a difference in developing countries. I gave examples of how GM crops are improving the lives of small-scale farmers in countries such as Indonesia, China, India, and various parts of Africa. Planting insect-resistant cotton gives farmers an increased yield of superior cotton and dramatically decreases the use of pesticides with concomitant improvement of the environment and the health of farmers and others who are exposed to such sprays. I discussed the type of developments that would specifically improve the lives of people in developing countries, such as ß-carotene enriched golden rice and cassava resistant to African Cassava Mosaic Virus. I then dealt with perceptions of lack of food safety associated with GM food and described the complexity of tests that GM crops and foods derived from them have to undergo in order to be declared safe. Finally, I discussed potential environmental impacts such as pollen transfer, horizontal gene transfer, and weediness.

Question time was lively, and I had an interesting interchange with the Ambassador from Zambia, a country that has refused maize food aid from the USA, as it might contain GM maize—this food aid refusal is in the face of major starvation among poor Zambians. The Ambassador accused scientists of not informing them of the true facts concerning GM food. Having myself visited Zambia recently and spoken with a number of scientists, and having talked to fellow scientists who have either also visited Zambia or have hosted Zambian politicians and scientists in the USA, I don't believe this to be the case. On the contrary, in my opinion the politicians' acceptance of the scientists' testimonies may be biased by political aims.

Judging from the questions we received, it was clear that many in the audience had very little knowledge of farming practice, for instance, that maize is a hybrid crop and unless farmers buy seed every year, whether GM or not, their yield will be severely diminished due to lack of hybrid vigor. I discussed open-pollinated varieties from which farmers could plant their own seed, but explained that these varieties would not be suitable for large scale cultivation and certainly not for export.

Quality is a combination of various desirable and economically significant attributes that make vegetables and fruits attractive and acceptable, such as size and shape, color, taste, flavor, gloss, presence of external and internal defects, texture, and softening. These attributes comprise complex parameters and are determined by a number of characteristics. For example, textural quality is determined, among other things, by fruit firmness or softness, fibrousness or toughness, succulence, and sensory qualities. A large number of cellular chemicals contribute to tomato aroma, while fruit taste is determined by acidity caused by organic acids and sugars. Thus, it is conceivable that each of these quality features is a result of highly regulated, multiple processes inherent in the commodity. Modern research has shown that most fruit quality attributes are controlled by sets of genes.

Many factors can negatively affect nutritive composition of a commodity, two of which are short shelf life and microbial infestation. Over-ripening, senescence, and physiological disorders, including chilling injury, are the major causes of post-harvest losses of fruit and vegetables. The genetic and biochemical bases of most of these processes remain to be explored. Losses during post-harvest storage are an economic drain and represent one of the greatest threats to a grower. For instance, tomato fruit decay during post-harvest results in a loss of 20% of marketed fruit, comprising the single greatest economic impact on tomato fruit commerce. Pathogen stress results in decay and development of off-flavor in fresh produce, rendering it unsuitable for consumption. To reduce the threatening impact of fruit rot, chemical fungicides are extensively applied, often on a weekly basis. Most chemicals used to control microbial infestation are toxic and contribute to an unfriendly environment.

Our focus is on using basic biology underlying fruit/vegetable development and ripening/senescence to develop novel, high nutritive-quality and stress-tolerant vegetable and fruit germplasm for the fresh market and processing industries. To reach these goals, we are applying an integrative approach comprised of biochemistry, molecular genetics, and biotechnology, using tomato as a fruit/vegetable model. The development of novel and improved vegetable crops using genetic engineering technology holds great promise. This molecular approach has, in principle, opened the entire living kingdom as a source of genes for introgression into established cultivars, and at the same time made possible rapid transfer of individual genes from the resource population to a particular cultivar, or for the reinsertion of native loci in increased copy number after laboratory manipulation. Biotechnology can be effectively applied in enhancing the nutritional quality of foods—for example, it is desirable to decrease the levels of toxic metabolites like allergens and to increase the levels of selected nutrients such as lycopene, polyamines, vitamin E, and glutathione, which work as anti-oxidants and decrease the risk of nutritional deficiencies.

Ripening of tomato fruit involves differentiation of chloroplast into chromoplasts, and most of the nutrients beneficial to human health—ß-carotene, vitamin E, and lycopene—accumulate in the chromoplasts. Careful genetic manipulation of processes or metabolites that can stabilize and maintain these chromoplasts in an anabolic state for a longer duration should allow development of transgenic plants with enhanced phytonutrient content. A major obstacle, however, in achieving these objectives is the limited basic information on genes of horticultural and economic importance, particularly since regulatory genes are expressed in low quantities, are highly regulated, and are expressed only in specific tissues at a specific time.

Plant growth, development, and senescence are regulated by a set of plant growth substances that function individually or in unison at specific stages in a plant's life. A set of these, such as auxins, gibberellins, cytokinins, and brassinosteroids, are generally considered as promoters of growth and development, while methyl jasmonate, abscisic acid, and ethylene promote senescence and cell death. In addition to these plant hormones, polyamines have been implicated in cell division, embryogenesis, root formation, floral initiation and development, fruit development and ripening, pollen tube growth, and senescence. However, direct evidence for any physiological role(s) polyamines may have in plant growth, development, and senescence is just emerging. Polyamines are considered anti-senescence in nature and have been implicated in regulating or limiting the action of the pro-senescence hormone ethylene.

Ethylene is synthesized from S-adenosylmethionine (SAM) by a sequential action of two key ethylene-biosynthesis enzymes, 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase. Ethylene is a simple, gaseous hydrocarbon that acts as a dominant hormone in post-harvest physiology and senescence of plant organs. The last century witnessed the cloning and characterization of several ripening-specific gene transcripts, genetic analysis of the various single-gene ripening mutants of tomato, the identification and cloning of genes encoding for each step in ethylene biosynthesis, and identification of ethylene receptors.¹ Genetic manipulation of one or more of these genes has contributed to the practical application of this knowledge to extend the shelf life of horticulturally-important crops.

Biosynthetic pathways for ethylene and polyamines in plants share SAM as a key intermediate. In this regard, it would appear that a plant cell has the potential to commit the flux of SAM either into polyamine biosynthesis, ethylene biosynthesis, or both.² Polyamines are ubiquitous in flowering plants and mainly comprise putrescine, cadaverine, 1,3-diaminopropane, spermidine, and spermine; however, other modified forms are also known in plants. Putrescine is formed from either arginine via an intermediate agmatine, a reaction catalyzed by arginine decarboxylase (ADC), or from ornithine by ornithine decarboxylase (ODC). Spermidine is synthesized from putrescine and the aminopropyl group donated by decarboxylated SAM, which is a product of SAM decarboxylation. In turn, spermidine incorporates another aminopropyl group (from decarboxylated SAM) to form spermine.³

To unambiguously demonstrate the role(s) of polyamines in the ripening process, whether through an effect on ethylene biosynthesis or ethylene action or other signals, my laboratory in collaboration with Prof. Avtar Handa of Purdue University (West Lafayette, Indiana) used a transgenic approach to accumulate polyamines at higher levels during ripening in tomato fruit. We reconstructed the yeast SAM decarboxylase (ySAMdc) gene⁴ by fusing it with a ripening-inducible promoter⁵ and introduced it into tomato plants using Agrobacterium-mediated gene transfer. Segregation analysis indicated Mendelian inheritance of the gene. Among four independent lines found homozygous with the introduced ySAMdc gene, two were fully characterized. The two transgenic lines produced fruit with several-fold increases in the levels of spermidine and spermine, while the wild type and azygous lines showed a consistent decrease during ripening in these polyamines. The accumulation of polyamines in the transgenic lines paralleled the accumulation of the ySAMdc transcripts. The effects of unusual accumulation of spermidine and spermine during ripening of the fruit resulted in delayed ripening of the fruit on the vine by two weeks, as compared to the fruit from the wild-type plants, and the juice quality was vastly improved.⁶ There was no marked change in the fruit yield between the lines.

Further, we found that the transgenic fruit we developed accumulated several-fold more of the carotenoid lycopene compared to the non-transformed fruit.⁶ Red tomato fruit color is primarily determined by lycopene content. Transformation of chloroplasts into chromoplasts during tomato fruit ripening leads to chlorophyll loss and increased content of carotenoids such as lycopene. Carotenoids are lipid soluble pigments found in chloroplast and chromoplast membranes.⁷ In addition to their role in photosynthetic reaction centers, they are essential for photoprotection. Moreover, they are also precursors of abscisic acid (ABA), a plant hormone that modulates developmental and stress processes. Polyamines are, among other processes, likely involved in stabilization of chromoplast function in plants.

Further investigation into this area should reveal novel regulation of plant metabolic processes, which may yet present us with new approaches to engineer metabolic pathways. These high polyamine-accumulating transgenic tomato lines are providing an excellent model system to investigate the role of polyamines in regulating gene expression, metabolism, growth, and development in plants and fruit ripening/senescence processes. For instance, we are investigating if polyamine accumulation results in changing the cellular redox systems due to their anti-oxidative nature, what processes are involved in polyamines-mediated stabilization of cellular membranes, which gene clusters are regulated by polyamines-mediated signaling, and how polyamines tilt the developmental balance towards anabolic from catabolic metabolism.

I thank my colleagues listed in the references for collaboration and stimulating research.

References

1. Fluhr R and Mattoo AK. 1996. Ethylene — biosynthesis and perception. Critical Reviews in Plant Science 15: 479-523.
2. Cassol T and Mattoo AK. 2002. Do polyamines and ethylene interact to regulate plant growth, development and senescence? In: Molecular and Cellular Biology: New Trends (P. Nath, A. Mattoo, S. Ranade and J. Weil, eds.), SMPS Publishers, Dehradun, India.
3. Cohen SS. 1998. A Guide to the Polyamines. Oxford Univ. Press, New York, pp. 595.
4. Kashiwagi K, Teneja SK, Liu TY, Tabor CW, and Tabor H. 1990. Spermidine biosynthesis in Saccharomyces cerevisiae. Biosynthesis and processing of a proenzyme form of S-adenosyl-methionine decarboxylase. Journal of Biological Chemistry 265: 22321-22328.
5. Giovannoni JJ, DellaPenna D, Bennett AB, and Fischer RL. 1989. Expression of a chimeric polygalacturonase gene in transgenic rin (ripening inhibitor) tomato results in polyuronide degradation but not fruit softening. The Plant Cell 1: 53-63.
6. Mehta RA, Cassol T, Li N, Ali N, Handa AK, and Mattoo AK. 2002. Engineered polyamine accumulation in tomato enhances phytonutrient content, juice quality, and vine life. Nature Biotechnology 20: 613-618.
7. Bartley GE and Scolnik PA. 1994. Molecular biology of carotenoid biosynthesis in plants. Annual Reviews of Plant Physiology and Plant Molecular Biology 45: 287-301.

THE FINGERS OF GENE REGULATION IN PLANTS
Roger N. Beachy and M. Isabel Ordiz

The machinery that controls gene expression in all organisms consists of a series of intracellular processes in which specific proteins, broadly referred to as transcription factors, play a significant role. Transcription factors have a modular structure: a DNA binding domain which targets the protein to specific DNA sequence(s); and a regulatory domain that acts to increase or decrease transcription of the gene(s) that contains the DNA binding domain.

Of all transcription factors, zinc finger-containing proteins are the most abundant in nature. It is estimated that 700 genes encode such factors in humans, while the model plant Arabidopsis thaliana contains about 85 genes of this class. Natural zinc finger proteins contain three zinc finger domains. Each single domain contains 30 amino acids that create a structure of two ß-sheets and one a-helix (bba), stabilized by hydrophobic interactions and the presence of a single zinc ion. The a-helix structure facilitates the interaction of the protein with three nucleotides (triplet). Polydactil six-zinc finger proteins that recognize eighteen consecutive nucleotides have been artificially engineered, thus creating a protein that is highly specific for 18 nucleotides.

By combining the binding characteristics of a synthetic six-zinc finger with a regulator domain, e.g., the herpes simplex virus VP16 activation domain, we explored the use of zinc finger technology in plants to control the expression of novel and endogenous genes.

In our experiments, we used a well-characterized six-zinc finger protein, 2C7-protein, and its DNA recognition sequence. To measure the interaction of the two elements, ß-glucuronidase reporter genes that contained the 2C7 DNA binding site were constructed and introduced to cells along with genes encoding the 2C7 proteins. In tobacco protoplasts, there was significant induction of reporter gene expression, subject to a variety of conditions. ¹ For example, the expression of the reporter gene was dependent on the distance between the 2C7 sites and TATA box of the reporter gene construct. The addition of the herpes simplex virus VP16 activation domain or a four times repeat of the minimal activation domain from VP16 (referred to as VP64) to the 2C7 protein increased expression of the reporter gene from 5 to 30 fold in tobacco protoplasts. In transgenic plants, expression of the reporter gene increased by as much as 450 fold.¹

In related experiments, the zinc finger activator protein was expressed from a phloem-specific promoter in transgenic plants that also contained a reporter gene construct. In these plants, the reporter gene was expressed only in the vascular tissues. Expression of the transgenes was stably inherited.¹

Stege and colleagues used a viral vector (based on tobacco mosaic virus) to test a variety of gene constructs in transient assays in BY-2 tobacco and maize protoplasts. The results of these studies indicate that the system can be useful in monocot as well as dicot cells. The use of C7 proteins that contain three-zinc fingers resulted in a higher level of gene activation (by about 4x) when compared with 2C7 six-zinc finger proteins. The authors suggested that the smaller DNA binding site might increase the occupancy of the protein on the C7 binding site.²

This paper also analyzed the effect of several known repressor domains (e.g., KRAB-A and SID) in this assay system. The KRAB-A domain of the Kruppel-associated box-A domain is known to act at the level of chromatin modification and remodeling; the mode of action of mSin3 (SID repressor domain) of the human MAD1 protein is not known. When attached to the six-zinc finger protein, the KRAB domain caused no specific repression of the reporter gene in the tobacco protoplasts; in contrast, the SID domain exhibited a 5-fold level of repression.

These data confirmed that it is possible to increase as well as decrease the expression of exogenous genes in plants using synthetic six-zinc finger proteins, thereby opening the possibility of controlling endogenous genes in plants.

To explore that possibility of regulating a native gene, Guan and colleagues developed a synthetic six-zinc finger protein that binds to a specific sequence upstream of the promoter of the Arabidopsis thaliana AP3 gene; AP3 controls floral organ identity.³ The effect of the zinc finger protein was tested with a reporter gene comprising the AP3 promoter and the ß-glucuronidase open reading frame. The positive or negative effect of the factor was mediated by the fusion of the zinc finger proteins with an activator (VP64) or repressor (SID) domain, under control of the AP1 promoter. The AP1 promoter is expressed throughout young floral primordia and in sepal and petal primordia in mature flowers. Plants co-transfected with the recognition sequence (AP3::GUS) and the activator protein (AP1::ZFP^AP3-VP64) showed expression of ß-glucuronidase in all primordia and in petals and sepals in later stages in a manner similar to the expression of AP1. Some plants had five or more petals and a reduced number of sepals; the stamens were normal and fertile.

In plants that contain the repressor protein AP1::Sid-ZFP^AP3 the ß-glucuronidase was expressed in stamens only. Petals in some flowers were absent and some were converted to sepals; these plants were fertile. When a constitutive promoter (UBQ3) was fused to the repressor protein, most of the plants were sterile, the petals were narrower and shorter than normal, and stamens were reduced in size with respect to the normal plant. The phenotype of these plants was similar to the phenotype previously described for ap3 and sap mutants. These characteristics were transmitted to the progeny in at least two consecutive generations,³ confirming that the zinc finger regulatory proteins can continue to exert their regulatory effect, as do endogenous regulatory genes.

The possibilities of using synthetic proteins to regulate at will the expression of endogenous genes will have many applications, including the study of gene function, in regulating the expression of novel genes and developing novel pathways for controlling expression of genes in plants, as well as other organisms. Clearly, the targeted application of this technology opens a wide range of possibilities in plant science and agricultural biotechnology.

References

1. Ordiz MI, Barbas CF III, and Beachy RN. 2002. Proc. Natl. Acad. Sci. USA 99: 13290-13295.
2. Stege J, Guan X, Ho T, Beachy RN, and Barbas CF III. 2002. Plant Journal (in press).
3. Guan X, Stege J, Kim M, Dahmani Z, Fan N, Heifetz P, Barbas CF III, and Briggs SP. 2002. Proc. Natl. Acad. Sci. USA 99: 13296-13301.

GENE MODIFICATION OF AN ENDOGENOUS GENE IN RICE PLANTS
Shigeru Iida and Rie Terada

ENHANCED RESISTANCE TO WATER DEFICIT STRESS IN TRANSGENIC TOMATOES
P. Janaki Krishna

Unlike migratory birds and other animals, most angiosperm plants cannot escape environmental stresses and hence have developed sophisticated mechanisms for defense. Research has revealed that many of the stresses, e.g., oxidative, salinity, drought, high light influence, water deficit etc., likely have interconnected defense responses. Therefore, examining individual stress responses independently may not provide complete information or afford optimal solutions. Fundamental research is beginning to unravel the molecular basis of abiotic stress resistance and has shown that transgenic plants exhibiting an elevation of enzymes that scavenge toxic oxygen species have increased abiotic stress tolerance. Stress responsive genes like Lea (late embryogenesis), rd (responsive to dehydration), and erd (early responsive to dehydration) have been isolated.

Many environmental stresses ultimately lead to dehydration in plants. A research team from the Institute of BioAgricultural Sciences (Taiwan) and the National Graduate Institute of Life Sciences (Taiwan) has developed transgenic tomato plants with an enhanced resistance to water deficit stress by over-expressing the CBF1 (C repeat/dehydration-responsive element binding factor 1) gene. CBF1 genes are regarded as `master switches' that in turn activate expression of COR genes, increasing freezing tolerance in transgenic plants in the absence of cold stimulation. A plasmid, containing a DNA cassette consisting of an Arabidopsis CBF1 cDNA driven by a 35S promoter, and nos terminator, ß-Glucuronidase (GUS), and NPTII reporter genes, was inserted into the tomato genome via Agrobacterium tumefaciens transformation.

Twenty-one unique transgenic tomato lines were obtained. Some progenies of transgenic plants were evaluated for resistance to water deficit stress. For the water deficit treatment, wild type and transgenic T1 plants were grown in the same pots at 24^oC, without water, for various periods, 0, 7, 14, 21 and 28 d, up to four weeks. The transgenic plants were more resistant to water deprivation than wild type—the water content of transgenic plants remained high during water deficit treatments, thereby reducing the tissue damage, while a marked reduction in water content was observed in wild type plants. The leaves of wild type plants became wilted and curled whereas transgenic plants did not. Less than 6% of the wild type survived after four weeks of water deficit. Gibberellic acid (GA3) pretreatment of both wild type and transgenic tomato plants had little or no impact on water deficit resistance.

Proline content was greater in transgenic plants than wild type under both normal and water deficit conditions. Absence of further elevation of Pro content during water deficit treatment indicates that over-expression of CBF1 under non-stress conditions protects plants from subsequent stresses. GA3 treatments did not affect Pro content in wild type and transgenic tomato plants. These results suggest that transgenic tomato plants possess an inherent resistance to water deficit conditions due to the expression of CBF1. However, phenotypically the transgenic tomato plants were shorter than wild type plants, with decreased fruit and seed numbers and fresh weights compared to those of wild type plants under normal growth conditions. Exogenous GA3 application improved fruit size, seed number, and fresh weight, though they did not reach the same levels as those of wild type plants. Results from additional chilling treatments conducted on transgenic tomato and wild type lines were similar to water deficit treatments—the transgenic plants were more resistant to chilling than wild type.

The researchers looked for CBF1 responsive genes using subtractive hybridization and isolated an upregulated CATALASE1 (CAT1) gene in the transgenic tomato plants. They further found that CAT1 expression and catalase activity were increased, and H₂0₂ concentration was reduced, in the transgenic tomato plants. The authors suggest that the upregulation of CAT1 might be due to the over-expression of CBF1. This phenomenon supports the hypothesis that activation of antioxidant genes confers the water deficit resistance seen in these transgenic tomato plants. The results further suggest that enhancement of the stress tolerance phenomenon in transgenic tomato plants might be conferred by multiple defense systems due to presence of CBF1, which acts as a master switch controlling various pathways responsive to stress.

The researchers are planning to isolate other upregulated genes like P5CS and others responsive to heterologous CBF1 that are important in transgenic tomato plants with water deficit resistance. Characterization of these stress responsive genes will contribute to an understanding of stress resistance and stress signal reduction pathways. In conclusion, the study suggests that heterologous CBF1 could improve environmental stress resistance in agriculturally important crop plants.

Reference

Hsieh TH, Lee JT, Charng YY, and Chan MT. 2002. Tomato plants ectopically expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress. Plant Physiology 130: 618-626.

Scientists have sought to develop a pig that has been genetically altered to allow pig-to-human organ transplants. Two approaches have been utilized recently. In the approach pursued by PPL Therapeutics (Blacksburg, VA) and the University of Missouri, the alpha galactosyl-transferase gene, which synthesizes an antigenic alpha galactose sugar on the cell surface, was deleted using a combination of gene targeting and nuclear transfer technology. In theory, pig cells unable to synthesize the alpha galactose sugar should be less antigenic to the human immune system. The other approach is to create transgenic pigs that express a human gene that suppresses the complement-mediated lysis of foreign cells.

In the October 29, 2002, issue of the Proceedings of the National Academy of Sciences, USA, a team of Italian researchers reports the production of transgenic pigs expressing human decay accelerating factor, a complement-inhibiting factor. This group used sperm-mediated gene transfer (SMGT) as a method of producing the transgenic pigs. Sperm-mediated gene transfer in mice was highly controversial when first published in 1989 because many groups subsequently had difficulty replicating the technique. However, since that time a number of reports have appeared showing that SMGT has been successfully used to generate transgenic fish, honeybee, sea urchin, chicken, and sheep.

Standard approaches to generating transgenic livestock include microinjection of DNA into fertilized eggs or transfer of foreign DNA into cultured cells followed by nuclear transfer into enucleated oocytes. In both cases, successes have been realized. However, with the microinjection method, the efficiency is low and with the nuclear transfer method the health of the cloned animals appears to be an issue.

To generate transgenic pigs, the Italian group first treated sperm from pre-selected boars with a DNA construct containing the human decay accelerating factor (hDAF) gene and then used this treated sperm for artificial insemination of eggs. Eight fertilizations in 15 gilts total were performed, resulting in the birth of 93 piglets, of which 53 (57%) carried the hDAF gene. Two fertilizations produced no transgenic piglets, while the other six resulted in 46-88% transgenic piglets. The transgene was stably integrated into the pig genome and was transmitted to progeny in a normal Mendelian fashion.

Human DAF mRNA and protein were detected in the transgenic pigs. In 34/53 (64%) of the transgenic piglets, the human DAF gene was expressed in all tissues examined. Expression of human DAF mRNA varied considerably between animals and varied in different tissues of the same animal. DAF mRNA expression was stable from birth to 6-12 months of age. Furthermore, human DAF protein was detected in the appropriate membrane fraction of transgenic pig heart and red blood cells.

The ability of human DAF to protect cells from lysis by antibodies and complement in fresh human serum was examined. Macrophages from transgenic pigs showed increased resistance to human serum compared with non-transgenic control pigs. With cells from control pigs, less than 10% of the cells survived treatment with human serum, whereas with cells from transgenic pigs, greater than 75% and often closer to 100% of the cells survived. Similar results were found when aortic endothelial cells from transgenic and control pigs were tested.

This report presents two significant findings: 1) the extremely high rate of transgenesis using sperm-mediated gene transfer; and 2) the generation of transgenic pigs expressing human decay accelerating factor. If the SMGT results can be verified, then this would represent a very simple, efficient, and cost effective method of producing transgenic animals. However, the one caveat is that this method allows for only the addition of genes, not the deletion of genes as has been accomplished with the alpha galactosidase gene. Nevertheless, the protection from complement-mediated lysis conferred upon the transgenic pig cells represents a promising step forward in the development of animal organs for human transplantation.

Lavitrano et al. 2002. Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hDAF) transgenic pigs for xenotransplantation. Proc. Natl. Acad. Sci. USA 99: 14230-14235.