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International audience; Background and aims: Apolipoprotein (apo) C1 is a 6.6 kDa protein associated with HDL and VLDL. ApoC1 alters triglyceride clearance, and it also favors cholesterol accumulation in HDL, especially by inhibiting CETP in human plasma. Apart from studies in mice, which lack CETP, the impact of apoC1 on atherosclerosis in animal models expressing CETP, like in humans, is not known. This study aimed at determining the net effect of human apoC1 on atherosclerosis in rabbits, a species with naturally high CETP activity but with endogenous apoC1 without CETP inhibitory potential.Methods: Rabbits expressing a human apoC1 transgene (HuApoC1Tg) were generated and displayed significant amounts of human apoC1 in plasma.Results: After cholesterol feeding, atherosclerosis lesions were significantly less extensive (-22%, p < 0.05) and HDL displayed a reduced ability to serve as CETP substrates (-25%, p < 0.05) in HuApoC1Tg rabbits than in WT littermates. It was associated with rises in plasma HDL cholesterol level and PON-1 activity, and a decrease in the plasma level of the lipid oxidation markers 12(S)-HODE and 8(S)HETE. In chow-fed animals, the level of HDL-cholesterol was also significantly higher in HuApoC1Tg than in WT animals (0.83 ± 0.11 versus 0.73 ± 0.11 mmol/L, respectively, p < 0.05), and it was associated with significantly lower CETP activity (cholesteryl ester transfer rate, -10%, p < 0.05; specific CETP activity, -14%, p < 0.05).Conclusions: Constitutive expression of fully functional human apoC1 in transgenic rabbit attenuates atherosclerosis. It was found to relate, at least in part, to the inhibition of plasma CETP activity and to alterations in plasma HDL.

Proteins are involved in a majority of the biochemical events that take place in all living organisms. Protein synthesis is directed by genes. All genes contain two major DNA regions. The region containing the genetic message proper (the 'coding region') is preceded by a regulatory region ('the promoter'), which determines when and in which organs a given gene must produce the corresponding protein. The techniques of genetic engineering allow the association of the coding region from one gene with the regulatory region from another gene. The expression of these recombinant genes may be achieved in cultured cells, in transgenic animals or in plants. This leads to the production of the corresponding proteins, including pharmaceutical proteins. Milk from transgenic animals is one potential source of pharmaceutical proteins. To achieve this, the promoters from milk protein genes are bound to DNA fragments containing the coding region of the genes of interest. The desired proteins are then taken from the milk and purified. Two human pharmaceutical proteins are on the market and about 20 projects are in development. One of the proteins produced in milk, antithrombin III, is an anticoagulant and the other, human C1-esterase inhibitor, is an anti-inflammatory. Several human proteins have been produced in the egg white of transgenic chickens and one has been approved by the United States Federal Drug Administration. This process has also been used to modify antibodies in cows. The genes that code for antibodies in the cow were deleted and replaced by human antibody genes. These cows, immunised by various antigens, then secreted purely human antibodies in their blood. Antibodies from such cows were able to attenuate the effects of Ebola virus in human patients.

International audience

Transgenesis is a potent method to generate new breeds of animals known as genetically modified animals (GMAs) or genetically engineered animals. Transgenesis includes two complementary gene modifications: foreign gene addition to animal genomes and gene replacement. The precise gene replacement may lead to the addition of an exogenous active gene (knock in) and the specific animal gene inactivation (knock out). The foreign genes may be transferred into pluripotent cells (mammalian embryonic stem or induced pluripotent stem), or into multipotent cells (chicken embryonic gonad cells) further used to generate chimeric transgenic animals. Somatic cells may also be used to generate transgenic clones. The rate of homologous recombination is markedly enhanced by cleaving locally the two genomic DNA strands using targeted nucleases. The cleaved DNA is then spontaneously and randomly repaired leading to targeted gene knock out. The generation of GMA including farm animals has thus become easier, cheaper, more precise, and safer. A number of vectors are being designed to optimize the expression of transgenes. Transgenic animals are being used to study gene mechanism and function, to adapt pig organs to be transferred to humans, to prepare recombinant pharmaceutical proteins in milk and egg white, and to improve breeding and food production.

The genetic modification of animals to obtain transgenic animals started in 1980. The first transgenic animals were mice, which are still the most frequently used transgenic species. About 20 transgenic species have been obtained and they are more or less currently used. Various methods are being implemented to transfer foreign genes to the different species. Transgenic animals are mostly used for basic research to study gene and biological functions. Transgenic animals may also be relevant models to study human and animal diseases as well as to test new medicaments. Transgenics may also be the source of organs and cells for humans as well as of medicaments. The impact of transgenesis to improve animals for food and feed production is still non-existent but is expected to become a reality in the coming months. A few transgenic pets have been obtained and commercialized. The use of transgenic animals raises some welfare problems and specific regulation has been defined by the EU. Transgenesis in animals al...

Given the history of GMO conflict and debate, the GM animal future is dependent on the response of the regulatory landscape and its associated range of interest groups at national, regional and international levels. Focusing on the EU and the USA, this article examines the likely form of that multi-level response, the increased role of cultural values, the contribution of new and existing interest groups and the consequent implications for the commercialization of both green and red GM animal biotechnology.

International audience; This paper reports our attempts to characterize transgene integration sites in transgenic mouse lines generated by the microinjection of large (from 30 to 145 kb) pig DNA fragments encompassing a mammary specific gene, the whey acidic protein gene (WAP). Among the various methods used, the thermal asymmetric interlaced (TAIL-) PCR method allowed us (1) to analyze transgene/genomic borders and internal concatamer junctions for eleven transgenic lines, (2) to obtain sequence information for seven borders, (3) to place three transgenes in the mouse genome, and (4) to obtain sequence data for seven transgene junctions in concatamers. Finally, we characterized various rearrangements in the borders and the inner parts of the transgene. The possibility of such complex rearrangements should be carefully considered when transgenic animals are produced with large genomic DNA fragments.

International audience; Proteins started being used as pharmaceuticals in the 1920s with insulin extracted from pig pancreas. In the early 1980s, human insulin was prepared in recombinant bacteria and it is now used by all patients suffering from diabetes. Several other proteins and particularly human growth hormone are also prepared from bacteria. This success was limited by the fact that bacteria cannot synthesize complex proteins such as monoclonal antibodies or coagulation blood factors which must be matured by post-translational modifications to be active or stable in vivo. These modifications include mainly folding, cleavage, subunit association, gamma-carboxylation and glycosylation. They can be fully achieved only in mammalian cells which can be cultured in fermentors at an industrial scale or used in living animals. Several transgenic animal species can produce recombinant proteins but presently two systems started being implemented. The first is milk from farm transgenic mammals which has been studied for 20 years and which allowed a protein, human antithrombin III, to receive the agreement from EMEA (European Agency for the Evaluation of Medicinal Products) to be put on the market in 2006. The second system is chicken egg white which recently became more attractive after essential improvement of the methods used to generate transgenic birds. Two monoclonal antibodies and human interferon-beta 1a could be recovered from chicken egg white. A broad variety of recombinant proteins were produced experimentally by these systems and a few others. This includes monoclonal antibodies, vaccines, blood factors, hormones, growth factors, cytokines, enzymes, milk proteins, collagen, fibrinogen and others. Although these tools have not yet been optimized and are still being improved, a new era in the production of recombinant pharmaceutical proteins was initiated in 1987 and became a reality in 2006. In the present review, the efficiency of the different animal systems to produce pharmaceutical proteins are described and compared to others including plants and micro-organisms.

The first transgenic animals, mice, were obtained in 1980. The techniques of gene transfer had to be adapted to obtain transgenic animals with an acceptable yield in about fifteen species. When the yield is low (low rate of random integration and targeted integration via homologous recombination), genetic modifications must be achieved in intermediate cells able to participate to the development of chimeric transgenic animals (ES cells, EG cells, iPS obtained by the dedifferentiation of somatic cells) or in somatic cells used as nuclear donor to generate transgenic clones. Various tools make possible a marked increase of homologous recombination efficiency (meganucleases and ZFN), or a gene inactivation at the genome level (direct or conditional knock out) or at the mRNA level (interfering RNAs). Vectors allow a more reliable transgene expression. Genetically modified animals are used mainly to obtain information on biological functions and human diseases. Transgenic animals produce recombinant pharmaceutical proteins in milk and soon in egg white. Pig organs adapted to be tolerated by patients might be tested in humans in five years. The projects based on the use of transgenesis to improve animal production are presently few. Transgenic salmon with accelerated growth might be on the market when their possible escape in oceans will be controlled.

Cloning via somatic cell nucleus transfer (SCNT) is a potential way for using validated genomes in farm animal breeding and to save endangered breeds or species. Although this technique is relatively inefficient and costly, it is envisaged to use it as an assisted reproduction technique. Despite numerous problems observed in the perinatal period, after some time clones appear normal although they may have kept some epigenetic modifications. Meat and milk from cattle and meat from pig clones and their offspring are substantially equivalent to conventional animals with no observed toxicity or allergenicity. Due to limited data, monitoring of clones and their offspring is recommended to detect whether there are unexpected long-term effects of cloning.

Distal control of the whey acidic protein (WAP) locus was studied using a transgenic approach. A series of pig genomic fragments encompassing increasing DNA lengths upstream of the mammary specific whey acidic protein (WAP) gene transcription start point (tsp) and 5 kb downstream were used for microinjection in mouse fertilized eggs. Our data pointed out three regions as potent regulators for WAP but not for RAMP3 gene expression (a non mammary-specific gene located 30 kb upstream of the WAP gene). WAP gene activating elements were present in the -80 kb to -30 kb and -145 kb to -130 kb regions whereas inhibitors were present in the -130 kb to -80 kb region. The stimulatory regions were characterized by peaks of histone H4 acetylation and a poor nucleosome occupancy in lactating sow mammary glands but not in liver. These data reveal for the first time the existence of several remote potent regulatory regions of the pig WAP gene.

International audience; Milk is a very abundant source of proteins for animal and human consumption. Milk composition can be modified using transgenesis, including exogenous gene addition and endogenous gene inactivation. The study of milk protein genes has provided researchers with regulatory regions capable of efficiently and specifically driving the expression of foreign genes in milk. The projects underway are aimed at modifying milk composition, improving its nutritional value, reducing mammary infections, providing consumers with antipathogen proteins and preparing purified recombinant proteins for pharmaceutical use. The present paper summarises the current progress in this field.

Animal imaging requires the use of reliable long-term fluorescence methods and technology. The application of confocal imaging to in vivo monitoring of transgene expression within internal organs and tissues has been limited by the accessibility to these sites. We aimed to test the feasibility of fibred confocal fluorescence microscopy (FCFM) to image in situ green fluorescent protein (GFP) in cells of living animals. We used transgenic rabbits expressing the enhanced GFP (eGFP) gene. Detailed tissue architecture and cell morphology were visualised and identified in situ by FCFM. Imaging of vasculature by using FCFM revealed a single blood vessel or vasculature network. We also used non-transgenic female rabbits mated with transgenic males to visualise eGFP expression in extra-foetal membranes and the placenta. Expression of the eGFP gene was confirmed by FCFM. This new imaging technology offers specific characteristics: a way to gain access to organs and tissues in vivo, sensitive detection of fluorescent signals, and cellular observations with rapid acquisition at near real time. It allows an accurate visualisation of tissue anatomical structure and cell morphology. FCFM is a promising technology to study biological processes in the natural physiological environment of living animals.

Rotaviruses are the main cause of infantile viral gastroenteritis worldwide leading to approximately 500,000 deaths each year mostly in the developing world. For unknown reasons, live attenuated viruses used in classical vaccine strategies were shown to be responsible for intussusception (a bowel obstruction). New strategies allowing production of safe recombinant non-replicating rotavirus candidate vaccine are thus clearly needed. In this study we utilized transgenic rabbit milk as a source of rotavirus antigens. Individual transgenic rabbit lines were able to produce several hundreds of micrograms per ml of secreted recombinant VP2 and VP6 proteins in their milk. Viral proteins expressed in our model were immunogenic and were shown to induce a significant reduction in viral antigen shedding after challenge with virulent rotavirus in the adult mouse model. To our knowledge, this is the first report of transgenic mammal bioreactors allowing the rapid co-production of two recombinant viral proteins in milk to be used as a vaccine.

The aim of the present study was to understand how the extracellular matrix (ECM) regulates at the gene level the prolactin (Prl)-induced signal transducer and activator of transcription 5 (STAT5)-dependent expression of the alpha s1-casein gene in mammary epithelial cells. CCAAT enhancer binding proteins (C/EBPs) are assumed regulators of beta-casein gene expression. Rabbit primary mammary cells express alpha s1-casein gene when cultured on collagen and not on plastic. Similar C/EBPbeta, C/EBPdelta, STAT5, and Prl-activated STAT5 were found under all culture conditions. Thus the ECM does not act through C/EBPs or STAT5. This was confirmed by transfections of rabbit primary mammary cells by a construct sensitive to ovine prolactin (oPrl) and ECM (6i TK luc) encompassing STAT5 and C/EBP binding sites. The mutation of C/EBPs binding sites showed that these sites were not mandatory for Prl-induced expression of the construct. Interestingly, chromatin immunoprecipitation by the anti-acetylhistone H4 antibody (ChIP) showed that the ECM (and not Prl) maintained a high amount of histone H4 acetylation upstream of the alpha s1-casein gene especially at the level of a distal Prl- and ECM-sensitive enhancer. Alpha6 integrin (a membrane receptor of laminin, the principal active component of the mammary ECM) was found at the surface of cells cultured on collagen but not on plastic. In cells cultured on collagen in the presence of anti-alpha6 integrin antibody, Prl-induced transcription of the endogenous alpha s1-casein gene was significantly reduced, without modifying C/EBPs and STAT5. Besides, histone H4 acetylation was reduced. Thus, we propose that the ECM regulates rabbit alpha s1-casein protein expression by local modification of chromatin structure, independently of STAT5 and C/EBPs.

The optimal utilization of energy substrates in muscle fibers is of primary importance for muscle contraction and whole body physiology. This study aimed to investigate the age-related changes in some indicators of glucose catabolism and fatty acid oxidation in muscles of growing rabbits. Longissimus lumborum (fast-twitch, LL) and semimembranosus proprius (slow-twitch, SMP) muscles were collected at 10 or 20 weeks of age ( n=6 per age). Glucose transporter GLUT4 content was investigated by immunoblot assay. Activity levels of five enzymes were measured: lactate dehydrogenase (LDH) and phosphofructokinase (PFK) for glycolysis; citrate synthase (CS), isocitrate dehydrogenase (ICDH) and -3-hydroxyacyl-coenzyme A dehydrogenase (HAD) for oxidation. Mitochondrial and peroxisomal oxidation rates were assessed on fresh homogenates using [1-14C]-oleate as substrate. At both ages, mitochondrial and peroxisomal oxidations rates, as well as activities of oxidative enzymes were higher in SMP than in LL. In both muscles, the apparent rate of fatty acid oxidation by the mitochondria did not differ between the two ages. However, a decrease in the activities of the three oxidative enzymes was observed in LL, whereas activities of CS and HAD and peroxisomal oxidation rate of oleate increased between the two ages in SMP muscle. In both muscles, LDH activity increased between 10 and 20 weeks, without variations in glucose uptake (GLUT4 transporter content) and in the first step of glucose utilization (PFK activity). In conclusion, mitochondrial oxidation rate of fatty acids and activities of selected mitochondrial enzymes were largely unrelated. Moreover, regulation of energy metabolism with advancing age differed between muscle types.

New transgenic pigs expressing combinations of regulators of complement activation and other molecules are needed to resist xenograft hyperacute rejection (HAR) and to further analyze and treat xenograft rejection. Double transgenic pigs for human CD55 (hCD55) and human CD59 (hCD59) using the promoter of the human elongation factor 1 alpha gene were generated, and their kidneys were transplanted into nonimmunosuppressed baboons. hCD55 and hCD59 were mainly expressed by the endothelial cells, and these cells showed increased resistance to complement-mediated lysis. Baboons receiving kidneys from hCD55hCD59 pigs survived for 5 and 6 days, and displayed alterations in coagulation. Thrombocytopenia and platelet microthrombi were present within the kidneys. Nontransgenic kidneys showed HAR in less than 2 days. Kidneys from pigs expressing hCD55hCD59 displayed protection against HAR in the absence of immunosuppression. Rejection was associated with coagulopathy leukocyte infiltration and a rebound of anti-alpha Gal antibodies.

Previous studies have shown that the 5'HS4 DNaseI hypersensitive site of the chicken beta-globin locus is endowed with classic insulator activities: (i) it blocks the interaction between promoter and enhancers when it is inserted between them (ii) it confers expression of integrated foreign genes independent of their position in the chromatin. The aim of this present work was to determine whether the 5'HS4 element was able to stimulate the expression level and/or to increase the expression frequency of a luc+ reporter gene controlled by the rabbit WAP gene promoter. Two constructs with 5'HS4 insulator (p5'HS4-WAPluc) or without (pWAPluc) were introduced in mouse fertilised oocytes. All transgenic lines containing the 5'HS4 element (six lines) expressed the transgene whereas only two out of eight lines harbouring the pWAP-luc construct expressed the transgene to a significant level. Moreover, the mean level of expression was seven times higher in p5'HS4WAP-luc lines than in pWAP-luc lines. Even all these benefits on transgene expression, the 5'HS4 element did not confer a copy-dependent expression, did not decrease the ectopic expression of the reporter gene and did not decrease the variability of expression. Thus, the 5'HS4 element does not have all the properties of a perfect insulator on a construct containing the luc+ reporter gene controlled by the rabbit WAP promoter.

Experimental data obtained in previous works have led to postulate that enhancers increase the frequency of action of a linked promoter in a given cell and may have some insulating effects. The multimerized rabbit alpha s1-casein gene enhancer, the 6i multimer, was added upstream of the rabbit whey acidic protein gene (WAP) promoter (-6,300; +28 bp) fused to the firefly luciferase (luc) gene (6i WAP-luc construct). The 6i multimer increased reporter gene expression in mouse mammary HC11 cells. In transgenic mice, a very weak but significant increase was also observed. More noticeable, no silent lines were found when the 6i multimer was associated to the WAP-luc construct. This reflects the fact that the 6i multimer tends to prevent the silencing of the WAP-luc construct. After addition of the 5'HS4 insulator region from the chicken beta-globin locus upstream of the 6i multimer, similar luciferase levels were measured in 6i WAP-luc and 5'HS4 WAP-luc transgenic mice. Our present data and previous ones, which show that the 6i multimer has no insulating activity on a TK gene promoter construct indicate that the insulating activity of the 6i multimer is construct-dependent and not amplified by the 5'HS4 insulator.

Background: Hyperacute rejection (HAR) of discordant xenografts in the pig-to-human combination can be prevented using tranplants expressing transgenic molecules that inhibit human complement. Hypodermin A (HA), a serine esterase that degrades C3, was tested in the guinea-pig-to-rat and in the pig-to-human combinations. Methods: Hypodermin A was tested in vitro, ex vivo, and in vivo models of HAR in the guinea-pig-to-rat combination. Hamster ovary cells (CHO) and a line of porcine aortic endothelial cells (PAEC11) were transfected with HA complementary DNA (cDNA). Results: The pattern of degradation of rat and human C3 by HA was different (multiple bands lower than 40 kDa) from the physiologic pattern observed after spontaneous degradation of rat C3 or physiologic activation of human C3. The CH50 activity in serum was significantly lower in rats treated with 3.2 mg HA/kg than in untreated rats (45 ± 16 U/ml vs. 700 ± 63 U/ml, P

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases affecting a wide range of mammalian species. They are caused by prions, a proteinaceous pathogen essentially composed of PrPSc, an abnormal isoform of the host encoded cellular prion protein PrPC. Constrained steric interactions between PrPSc and PrPC are thought to provide prions with species specificity, and to control cross-species transmission into other host populations, including humans. Transgenetic expression of foreign PrP genes has been successfully and widely used to overcome the recognized resistance of mouse to foreign TSE sources. Rabbit is one of the species that exhibit a pronounced resistance to TSEs. Most attempts to infect experimentally rabbit have failed, except after inoculation with cell-free generated rabbit prions. To gain insights on the molecular determinants of the relative resistance of rabbits to prions, we generated transgenic rabbits expressing the susceptible V136R154Q171 allele of the ovine PRNP gene on a rabbit wild type PRNP New Zealand background and assessed their experimental susceptibility to scrapie prions. All transgenic animals developed a typical TSE 6–8 months after intracerebral inoculation, whereas wild type rabbits remained healthy more than 700 days after inoculation. Despite the endogenous presence of rabbit PrPC, only ovine PrPSc was detectable in the brains of diseased animals. Collectively these data indicate that the low susceptibility of rabbits to prion infection is not enciphered within their non-PrP genetic background., Author Summary Prions are infectious pathogens causing irremediably fatal neurodegenerative diseases in human and in farmed or wild animals. They are formed from abnormally folded assemblies (PrPSc) of the host-encoded prion protein (PrPC). Different PrPSc conformational variants or ‘strains’ can propagate in the same host, giving distinct biological phenotypes. Like other pathogens, prions can transmit from one species to another, representing a zoonotic risk. A barrier, commonly referred to as “species barrier”, limits prion cross-species transmission. This barrier is supposed to reflect the steric incompatibility between invading prion PrPSc and PrPC of the infected host. Rabbit is one of the species that exhibit a pronounced resistance to prions. To gain insights on the molecular determinants of the relative resistance of this species to prions, we generated transgenic rabbits expressing sheep PrPC and assessed their experimental susceptibility to sheep scrapie prions, as routinely done with transgenic mouse models of prion disease. All transgenic rabbits developed a typical prion disease within 200 days, whereas wild type rabbits remained healthy more than 700 days after inoculation. These data indicate that the low susceptibility of rabbits to prion infection is not enciphered within their non-PrP genetic background.

Transgenic animals have been used for years to study gene function and to create models for the study of human diseases. This approach has become still more justified after the complete sequencing of several genomes. Transgenic animals are ready to become industrial bioreactors for the preparation of pharmaceuticals in milk and probably in the future in egg white. Improvement of animal production by transgenesis is still in infancy. Despite its intensive use, animal transgenesis is still suffering from technical limitations. The generation of transgenics has recently become easier or possible for different species thanks to the use of transposons or retrovirus, to incubation of sperm which DNA followed by fertilization by intracellular sperm injection or not and to the use of the cloning technique using somatic cells in which genes have been added or inactivated. The Cre-LoxP system is more and more used to withdraw a given sequence from the genome or to target the integration of a foreign DNA. The tetracycline system has been improved and can more and more frequently be used to obtain faithful expression of transgenes. Several tools: RNA forming a triple helix with DNA, antisense RNA including double strand RNA inducing RNA interference and ribozymes, and also expression of proteins having a negative transdominant effect, are tentatively being improved to inhibit specifically the expression of host or viral genes. All these techniques are expected to offer experimenters new and more precise models to study gene function even in large animals. Improvement of breeding by transgenesis has become more plausible including through the precise allele replacement in farm animals.

Transgenesis consists of introducing stably a foreign genetic information into the genome of a multicellular organism. These techniques used for the first time in 1980 for the animals and 1983 for plants have multiple applications of which many are directly or not related to medicine. Transgenesis has become one of the essential tools to study the role of genes in the control of biological functions. This approach is logically accompanied by the generation of transgenic animal lines for the study of human diseases and the test of new pharmaceuticals. Milk from transgenic animals as well as leaves and seeds from transgenic plants are ready to become an essential source of proteins having essential therapeutical effects. Genetically modified pigs are expected to be resistant to rejection mechanisms to become the source of organs or cells for patients. The application of transgenesis in agriculture and breeding is still in infancy. It may contribute quite significantly to provide human communities with food in sufficient amount, having improved nutritional properties and produced using a milder and less polluting methods.

The improvement of animal production by transgenesis is expected to be complementary to other breeding techniques including genetic selection (by conventional methods and using genetic markers), feed optimization, control of reproduction and struggle against diseases. It may introduce new and efficient methods to solve various problems as is already the case for plants. The success of transgenesis for animal production has been limited for 15 years by the difficulty and the cost to generate transgenic farm animals. Gene addition can be achieved by DNA microinjection into one-cell embryos in rabbit and pig. In ruminants, gene addition can be achieved by classical transfection in fetal cells further used to generate cloned transgenic animals. The cloning techniques have been used successfully to replace gene by homologous recombination in sheep and can be extended to goat, cow, pig and expectedly in rabbit. This may lead to precise gene inactivation or allele replacement. In birds, several techniques can be used to generate transgenics. None of these techniques can presently be used routinely to improve breeding. Transgenesis in farm animals is expected to protect animals against diseases, to reduce rejection of pollutants, to optimize digestion, to improve growth and fertility, to optimize meat and milk composition, etc… The production of pharmaceutical proteins in transgenic animals and the use of pig organs for xenografting are not expected to have any significant direct impact on animal production.

International audience; The preparation of recombinant pharmaceutical proteins is one of the major challenges of biotechnology. Mammalian cells are required for a number of proteins which must be modified posttranscriptionally. Animal cell lines cultured in fermentors are presently the major source of complex proteins. The milk of transgenic animals proved to be a possible source of pharmaceutical proteins and one of them, human antithrombin III, has been approved by the EU (EMEA) and US (FDA) medicament agencies. Several species are being implemented for this purpose. Rabbits are one of these species. It offers several advantages: low cost to produce transgenic founders, rapid reproduction, easy and cheap scaling up, easy breeding in pathogen-free conditions and insensitivity to prion diseases. Rabbits are thus an efficient tool to prepare several kilograms of a recombinant protein per year.

The whey acidic protein (WAP) is the major whey protein of rodent, rabbit and camel. Recently, it was identified in the milk of swine ( Simpson et al., 1998 . J. Mol. Endocrinol. 20, 27–35). In this paper, the cloning of the pig WAP cDNA and of bacterial artificial chromosome (BAC) construct containing the entire porcine WAP gene is reported. The comparison of the coding sequence of the pig WAP gene to rodent or lagomorph WAP sequence already published demonstrated that only exon sequences are partially conserved. The porcine WAP gene was localized on the subtelomeric region of the chromosome 18. The estimation of the expression of the swine WAP gene in the mammary gland from lactating animals revealed a high level of expression. In order to compare the expression level of the porcine WAP gene from the large genomic fragment which contained 70 kb downstream and 50 kb upstream the pig WAP gene or the smaller one (1 kb downstream and 2.4 kb upstream), these two genomic fragments were transfected in HC11 cell line. The BAC construct was expressed 15 times higher than the plasmid when reported to the integrated copy number. This report suggests that the HC11 cell line is a useful tool to identify the regulatory sequences of milk protein genes.

The IRES from poliovirus and from encephalomyocarditis virus (EMCV) added between the cap and the AUG initiator codon were strong inhibitors of chloramphenicol acetyltransferase gene expression in three different cell types. The poliovirus IRES also inhibited bGH (bovine growth hormone) cDNA expression in the HC11 mammary cell line when added between the rabbit whey acidic gene promoter and the cDNA whereas the HTLV-1 IRES showed a stimulatory effect in the same situation. RNA stem loops were added before HTLV-1 (SUR) and the BiP (Immunoglobulin heavy-chain Binding Protein) IRESs followed by the firefly luciferase gene under the control of Rous sarcoma virus (RSV) promoter. The RNA loops abolished the expression of the reporter gene almost completely. These data suggest that the different IRESs may favour or inhibit translation of monocistronic mRNA.

The production of recombinant proteins is one of the major successes of biotechnology. Animal cells are required to synthesize proteins with the appropriate post-translational modifications. Transgenic animals are being used for this purpose. Milk, egg white, blood, urine, seminal plasma and silk worm cocoon from transgenic animals are candidates to be the source of recombinant proteins at an industrial scale. Although the first recombinant protein produced by transgenic animals is expected to be in the market in 2000, a certain number of technical problems remain to be solved before the various systems are optimized. Although the generation of transgenic farm animals has become recently easier mainly with the technique of animal cloning using transfected somatic cells as nuclear donor, this point remains a limitation as far as cost is concerned. Numerous experiments carried out for the last 15 years have shown that the expression of the transgene is predictable only to a limited extent. This is clearly due to the fact that the expression vectors are not constructed in an appropriate manner. This undoubtedly comes from the fact that all the signals contained in genes have not yet been identified. Gene constructions thus result sometime in poorly functional expression vectors. One possibility consists in using long genomic DNA fragments contained in YAC or BAC vectors. The other relies on the identification of the major important elements required to obtain a satisfactory transgene expression. These elements include essentially gene insulators, chromatin openers, matrix attached regions, enhancers and introns. A certain number of proteins having complex structures (formed by several subunits, being glycosylated, cleaved, carboxylated...) have been obtained at levels sufficient for an industrial exploitation. In other cases, the mammary cellular machinery seems insufficient to promote all the post-translational modifications. The addition of genes coding for enzymes involved in protein maturation has been envisaged and successfully performed in one case. Furin gene expressed specifically in the mammary gland proved to able to cleave native human protein C with good efficiency. In a certain number of cases, the recombinant proteins produced in milk have deleterious effects on the mammary gland function or in the animals themselves. This comes independently from ectopic expression of the transgenes and from the transfer of the recombinant proteins from milk to blood. One possibility to eliminate or reduce these side-effects may be to use systems inducible by an exogenous molecule such as tetracycline allowing the transgene to be expressed only during lactation and strictly in the mammary gland. The purification of recombinant proteins from milk is generally not particularly difficult. This may not be the case, however, when the endogenous proteins such as serum albumin or antibodies are abundantly present in milk. This problem may be still more crucial if proteins are produced in blood. Among the biological contaminants potentially present in the recombinant proteins prepared from transgenic animals, prions are certainly those raising the major concern. The selection of animals chosen to generate transgenics on one hand and the elimination of the potentially contaminated animals, thanks to recently defined quite sensitive tests may reduce the risk to an extremely low level. The available techniques to produce pharmaceutical proteins in milk can be used as well to optimize milk composition of farm animals, to add nutriceuticals in milk and potentially to reduce or even eliminate some mammary infectious diseases.

Growing and differentiating keratinocytes maintain the epidermal barrier. This is partly controlled by growth factors and hormones. Prolactin (PRL) is named after its hormonal role in mammals during lactation, but is found in all vertebrates where PRL exerts various effects. In serum-free keratinocyte cultures, PRL was thought to be the factor responsible for the proliferative effect of bovine pituitary extract. Here, we evaluated PRL as a clonogenic factor for keratinocytes and found no mitogenic activity. Studying the expression of the PRL receptor by keratinocytes, we found the receptor upregulated only after culture confluence, in differentiating keratinocytes, but we were unable to detect any cellular response to PRL. The hormone does not alter the gene expression of either early (suprabasal keratin) or late (involucrin) differentiation markers by keratinocytes. Accordingly, no activation of the transcription factor Stat5 by PRL can be detected in keratinocytes, Stat5 being nevertheless detected by Western blot.

Whatever its field of application, animal transgenesis aims at a high level of reproducible and stable transgene expression. In the case of xenotransplantation, prevention of hyperacute rejection of grafts of animal origin requires the use of organs expressing human inhibitors of complement activation such as CD55 (DAF) and CD59. Pigs transgenic for these molecules have been produced, but with low and variable levels of expression. In order to improve cDNA expression, a vector containing the 5'HS4 region from the LCR of the chicken beta-globin locus and the promoter and the first intron from the human EF1 alpha gene, was used to co-express human CD55 and CD59 cDNAs in transgenic rabbits. The transgenic lines with the 5'HS4 region displayed dramatically enhanced CD55 and CD59 mRNA concentrations in brain, heart, kidney, liver, lung, muscle, spleen and aortic endothelial cells in comparison with the transgenic lines without the 5'HS4 region. In the absence of the 5'HS4 region, only some of the transgenic lines displayed specific mRNAs and at low levels. Human CD55 and CD59 proteins were detectable in mononuclear cells from transgenic rabbits although at a lower level than in human mononuclear cells. On the other hand, primary aortic endothelial cells from a bi-transgenic line were very efficiently protected in vitro against human complement-dependent lysis. Transgenic rabbits harbouring the two human inhibitors of complement activation, CD55 and CD59, can therefore be used as new models in xenotransplantation. Moreover, the vector containing the 5'HS4 region from the LCR of the chicken beta-globin locus seems appropriate not only for xenotransplantation but also for any other studies involving transgenic animals in which cDNAs have to be expressed at a high level in all cell types.

IRESs are known to recruit ribosomes directly, without a previous scanning of untranslated region of mRNA by the ribosomes. IRESs have been found in a number of viral and cellular mRNAs. Experimentally, IRESs are commonly used to direct the expression of the second cistrons of bicistronic mRNAs. The mechanism of action of IRESs is not fully understood and a certain number of laboratories were not successful in using them in a reliable manner. Three observations done in our laboratory suggested that IRESs might not work as functionally as it was generally believed. Stem loops added before IRESs inhibited mRNA translation. When added into bicistronic mRNAs, IRESs initiated translation of the second cistrons efficiently only when the intercistronic region contained about 80 nucleotides, and they did not work any more effectively with intercistronic regions containing at least 300-400 nucleotides. Conversely, IRESs inserted at any position into the coding region of a cistron interrupted its translation and initiated translation of the following cistron. The first two data are hardly compatible with the idea that IRESs are able to recruit ribosomes without using the classical scanning mechanism. IRESs are highly structured and cannot be scanned by the 40S ribosomal subunit. We suggest that IRESs are short-circuited and are essentially potent stimulators favoring translation in particular physiological situations.

The transferrin gene is expressed in all mammals, mainly in the liver. A rabbit genomic library was screened using cDNA probes, and 8 kb of 5′ flanking sequence of the rabbit transferrin gene was cloned upstream of the cat reporter gene. The first 200 nucleotides of this promoter were sequenced. The rabbit transferrin promoter is highly homologous to the human and murine ones. Its functional activity was tested in the human hepatic cell line HepG2. Using transitory transfections in these cells, a proximal positive region, a negative region and a distal positive region located between −3.6 and −4.0 kb were identified. This distal positive region sequence is highly conserved with the the human gene enhancer sequence, and contains an HNF3 α binding site, the mutation of which totally abolished its effect in HepG2 and HuH7 cell lines. The rabbit transferrin 5′ flanking sequence thus shows a promoter organization very similar to that of the human gene, and the HNF3 α binding site in the distal positive region presents the same functional importance in both genes.

The establishment of cell lines allows reproductible in vitro studies that would be far more difficult to perform using primary cells that rapidly undergo phenotypical alterations in culture. The purpose of this work was to establish an endothelial cell line appropriate for in vitro study of endothelial cell activation during xenograft rejection. Porcine aortic endothelial cells were transfected with the early region of SV40 and selected on the basis of morphological, phenotypical, and functional features. By light and electron microscopy, the porcine aortic endothelial cell line (PAEC11) and primary cells were similar except that PAEC11 was slightly smaller. PAEC11 displayed endothelial cell characteristics since it endocytosed acetylated low density lipoproteins, produced von Willebrand factor, and expressed E-selectin. Human natural antibodies bound to the same xenoantigens on PAEC11 and primary cells. That binding was followed by human complement activation and cell lysis. In addition, PAEC11 was found appropriate for genetic engineering since it could be transfected with a plasmid encoding a foreign gene. Therefore, this cell line should be a useful model for in vitro study of endothelial cell function in general and human-to-swine xenograft rejection in particular.

To prevent transgene silencing and unspecific expression, vectors must contain either multiple and appropriate regulators or long genomic DNA fragments containing enhancers and insulators. Alternatively, gene constructs may be targeted to chosen genome sites using homologous recombination. RNAi may be used to knock down endogenous or viral genes. Transgenes may be designed to be controlled by exogenous regulators improving the relevance of the mouse models.

Purpose – Food products developed using genetically modified (GM) animals may soon be introduced in Europe and beyond. Their successful commercialisation depends on consumer acceptance, and so it is timely to review the existing literature in this respect. The paper aims to discuss these issues. Design/methodology/approach – A systematic review identified 42 English language peer reviewed papers assessing public opinion of GM animals associated with food production. Thematic analysis was applied to the results to identify and explain consumer attitudes. Findings – Publication peaked in 2004, and declined thereafter. European consumers were less accepting of GM animal technology than the US and Asian consumers, although the latter reported more ethical concern. Risk and benefit perceptions, ethical concerns (e.g. related to animal welfare) may explain negative consumer attitudes towards animals in food production. Research limitations/implications – There is a lack of data on consumer attitudes to GM animals applied to food production, in particular in relation to consumers in emerging economies and developing countries. This is problematic as applications of GM animal products are about to enter the market. Practical implications – There is a need to track changes in public opinion as GM food production animals are further developed. The introduction and commercialisation of applications with specific characteristics may further shape consumer attitudes. Social implications – Methods need to be developed to involve consumers and other stakeholders in shaping future applications of agri-food applications of GM animals. Originality/value – The review collates existing quantitative and qualitative knowledge regarding the drivers of consumer attitudes towards GM animals used in food production using systematic review methodology.

Expression of human extracellular superoxide dismutase (EC-SOD), a glycosylated, tetrameric metalloprotein, was targeted to the lactating mammary gland of transgenic rabbits. Efficient expression of the recombinant whey acidic protein/ec-sod gene was achieved and up to 3 mg ml-1 of the enzyme was secreted into the milk. Rabbit milk-produced recombinant EC-SOD was primarily found in the whey and purified by a two-step chromatographic method. To evaluate the rabbit milk-produced human EC-SOD, comparisons with native and Chinese hamster ovary cell (CHO)-produced EC-SOD were performed. All proteins were tetrameric and N-glycosylated. The behaviour on SDS-PAGE and size-exclusion chromatography indicated that the masses, and thereby the extent of post-translational modification of the proteins was similar. The monosaccharide composition of both recombinant EC-SOD variants was analysed and indicated similarities in the attached N-glycans on the two proteins. Furthermore, the peptide maps of the three EC-SOD variants revealed that all proteins had similar polypeptide backbones.