François Pothier

Making recombinant proteins in animals – different systems, different applications

Transgenic animal bioreactors represent a powerful tool to address the growing need for therapeutic recombinant proteins. The ability of transgenic animals to produce complex, biologically active recombinant proteins in an efficient and economic manner has stimulated a great deal of interest in this area. As a result, genetically modified animals of several species, expressing foreign proteins in various tissues, are currently being developed. However, the generation of transgenic animals is a cumbersome process and remains problematic in the application of this technology. The advantages and disadvantages of different transgenic systems in relation to other bioreactor systems are discussed.

Electroporation of bovine spermatozoa to carry DNA containing highly repetitive sequences into oocytes and detection of homologous recombination events

There are several methods of modifying bovine genomes. Pronuclear microinjection is more widely used but it is still to be improved. Searches for alternatives have lead to the development of new methods including SMGT (Sperm Mediated Gene Transfer), in which live spermatozoa are used as vehicles for DNA delivery during in vitro fertilization. In previous studies, we presented evidence that a highly repetitive Alu‐like repeat favours transgenesis by homologous recombination (HR). Up to 60% integration via HR was obtained following pronuclear microinjection of a Pst1 β‐actin GFP DNA construction. In the present study, we show that HR‐mediated integration is also possible using SMGT, since bovine spermatozoa electroporated with the same DNA construct are able to transfer it to a high proportion of embryos obtained by in vitro fertilization. Swim‐up selected bovine spermatozoa were mixed with the Pst1 β‐actin GFP construct (6 × 106 spermatozoids were incubated with 600 ng of μDNA), submitted or not to electroporation (300 V, 25 F) and treated or not with DNase I. The process of electroporation itself did not affect in vitro embryonic development. However, oocytes fertilized with electroporated DNA‐treated spermatozoa developed beyond the 16‐cell stage in proportions that were significantly lower (27% with Pst1 β‐actin GFP and 34% with β‐actin GFP) compared to the control without DNA (44%). On the other side, the use of electroporation significantly increased the uptake of DNA. The number of homologous recombination events detected by PCR went from 3.5% without electroporation to 46.5% after electroporation. In conclusion, our results confirm that spermatozoa electroporation combined with homologous recombination in a highly repetitive Pst1 sequence is a feasible method to obtain transgenic bovine embryos. Mol. Reprod. Dev. 57:338–345, 2000.

Transgenic Glutathione Peroxidase Mouse Models for Neuroprotection Studiesa

Seleno-glutathione peroxidase (GSHPx) is considered to be the major enzymatic activity in charge of removing excess cytosolic and mitochondrial H2O2 in most tissues including brain. Intracellular GSHPx activity is therefore hypothesized to be one important factor that contributes to minimize hydroxyl radical formation via Fenton-type reactions. An animal model was developed to challenge this hypothesis in vivo and evaluate the role of GSHPx in hydroperoxide metabolism and oxidative stress homeostasis. Three lines of transgenic mice, homozygous for the integration of 1 to 3 GSHPx transgene copies, have been generated. The transgene was placed under transcriptional control of a metallothionein promoter (hMT-IIA). This promoter was chosen because metallothionein expression, normally low in most tissues, can be induced by several inflammatory cytokines, protein kinase C activators, and stress agents including heavy metals. The data reported here provide information on the constitutive expression of GSHPx mRNA and enzyme in various brain regions of healthy untreated adult tg-MT-GPx mice. Northern and/or Western analysis indicated that transgenic GSHPx was expressed constitutively in all brain regions investigated in tg-MT-GPx-6 mice, including the cerebral cortex, brainstem, hippothalamus, cerebellum, substantia nigra, and striatum. Similar results were obtained with the two other transgenic lines, tg-MT-GPx-11 and -13. Depending on the brain region, the GSHPx immunoreactivity detected in tissue extracts with an immunoaffinity-purified polyclonal antibody was about 2- to 5-fold stronger in transgenic extracts than in their non-tg counterparts (western blots). In contrast, the corresponding increase in GSHPx activity measured in these extracts was smaller, for example, about 1.5-fold in transgenic mesencephalon. Immunocytochemical data indicated that GSHPx-like staining was distinctly more intense in transgenic midbrain brain sections than in corresponding non-tg sections. Interestingly, only a subset of the cells displayed higher density staining that most likely reflects increased amounts of GSHPx protein. This observation suggests that the stained cells, not yet identified, may have larger GSHPx activity increments than the cell-average increments measured in tissue extracts. Current work is in progress to determine whether transgenic GSHPx expression may be induced by inflammatory processes or perturbations of heavy metal metabolism.

Seminal vesicle production and secretion of growth hormone into seminal fluid

Production of foreign proteins in the tissues of transgenic animals represents an efficient and economical method of producing therapeutic and pharmaceutical proteins. In this study, we demonstrate that the mouse P12 gene promoter specific to the male accessory sex gland can be used to generate transgenic mice that express human growth hormone (hGH) in their seminal vesicle epithelium. The hGH is secreted into the ejaculated seminal fluids with the seminal vesicle lumen contents containing concentrations of up to 0.5 mg/ml. As semen is a body fluid that can be collected easily on a continuous basis, the production of transgenic animals expressing pharmaceutical proteins into their seminal fluid could prove to be a viable alternative to use of the mammary gland as a bioreactor.

Transplanted Bone Marrow Cells Do Not Provide New Oocytes But Rescue Fertility in Female Mice Following Treatment With Chemotherapeutic Agents

It is generally accepted that mammalian females are born with a finite pool of oocytes and that this is the sole source of ovules throughout the reproductive life of the adult. This dogma was shaken in 2003 when researchers showed that the oocyte stock might be renewable in adult mammals. It has been proposed that hematopoietic stem cells might be a source of new oocytes. These discoveries have puzzled many researchers and remain controversial. In our study, we attempted to determine if transplanted bone marrow cells could provide new oocytes in PU.1 mice and in severe combined immunodeficiency (SCID) mice after treatment with chemotherapeutic agents. We also examined the possibility that grafted bovine embryonic ovarian cortex might provide an environment favoring such a response. We found no evidence that transplanted bone marrow cells provide new fertilizable oocytes in PU.1 mice, in SCID mice treated with chemotherapeutic agents, or with bovine embryonic ovarian tissue grafts. However, transplanted bone marrow cells have improved the fertility of SCID mice previously treated with chemotherapeutic agents. These data suggest that bone marrow cells cannot provide new oocytes but can positively influence ovarian physiology to improve the fertility of mice previously treated with chemotherapeutic agents.

Tissue specificity and ontogeny of lactate dehydrogenase in snow crab, Chionoecetes opilio (Brachyura, Majidae)

Abstract In the snow crab, Chionoecetes opilio (Brachyura: Majidae), LDH is present in rive isozymic forms, as revealed by the rive coloration zones in zymograms. This is characteristic of a tetrameric LDH enzyme formed by the random association between two distinct sub-units. Isozymes of C. opilio LDH show tissue specificity. No LDH activity is detected in the hepatopancreas. The LDH-B∗ locus is expressed in all other tissues examined, except the eye, in which LDH-A∗ is the only locus detected. Both loci are expressed in nervous, midgut and leg muscle tissues, the last showing individual variability. At least among males, the LDH-B∗ locus is expressed in leg muscle tissue at every developmental stage, while LDH-A∗ locus expression is initiated only in the VII th post-larval stage, just before the onset of gametogenesis. Results suggest that LDH sub-units in C. opilio have distinct physiological roles. This is very well known in vertebrates, but is virtually undocumented in invertebrates.

Use of bovine satellite sequences to increase transgene integration by homologous recombination in bovine embryos

Homologous recombination (HR) has proven to be functional in mammalian embryos. The efficiency of the HR process was tested in bovine zygotes in an attempt to increase the frequency of transgene integration using different lengths of a bovine satellite (BS) DNA flanking both ends of a neo gene marker (called BS500, BS250, and BS50) and neo alone as a control. Pronuclear microinjection at 16–19 hr post insemination (hpi) of the BS500, BS250, BS50 or neo fragments at a concentration of 1 ng/μl resulted in an increasingly negative effect on embryo development. Therefore all microinjections were performed at a single molecular concentration (320 × 106 molecules/μl). After microinjection, the embryos were allowed to develop for 6 days followed by morphological and PCR analysis. The HR event was detected by PCR in 13 of the 26 embryos (43%) that developed beyond the 12‐cell stage, 7/22 (31%), 9/27 (33%), and 0/25 (0%) with the BS500, BS250, BS50, and neo constructs respectively. The length of BS homology had no effect on transgene integration. However, embryos injected with BS neo constructs had significantly lower development rates than neo injected zygotes (17% more than 16 cells for BS500; 14% for BS250; 16% for BS50 compared to 32% for neo, P < 0.05, 6 replicates). These results demonstrate that BS sequences have a negative effect on embryo development and survival regardless of the amount of DNA injected. The use of HR with highly repetitive genomic sequences is therefore a feasible procedure to produce transgenic bovine embryos. Mol. Reprod. Dev. 53:1–7, 1999.

Developmental potential of early bovine zygotes submitted to centrifugation and microinjection following in vitro maturation of oocytes

This study was designed to investigate the potential use of in vitro matured, in vitro fertilized bovine zygotes for producing transgenic calves by microinjection of foreign DNA. In Experiment 1, the effect of centrifugation (4 min, 15,000 x g, 20 degrees C) on in vitro derived bovine zygotes was evaluated. In vitro development from 2 to 8 cells was not affected (80 vs 78%) when control zygotes (n = 211) were compared with zygotes treated (n = 210) 18 h post insemination. In Experiment 2, the influence of the centrifugation alone on the developmental potential of embryos was evaluated in rabbit oviducts for 120 h. The percentage of control and treated zygotes that developed to 1, 2 to 8, 8 to 32 and more than 32 cells were 7, 54, 10 and 10% vs 7, 40, 11 and 10%, respectively. In Experiment 3, the effect of pronuclear injection with plasmid containing CRF (corticotropin releasing factor) gene or pOCAT 330 Delta 1 plasmids; 2 microg/ml in Tris 10 mM, EDTA 0.2 mM, 18 to 20 h post insemination was evaluated by in vivo development in the rabbit oviduct. The embryos submitted only to centrifugation and vortexing resulted in a morula-blastocyst (> 32 cells) rate of 25% (n = 226) compared with the injected zygotes of which only 5% (n = 206) achieved the same stage. We conclude that in vitro produced bovine zygotes have a reduced developmental potential following microinjection, and this effect is not due to the centrifugation process.

The effects of vasopressin and related peptides on osmoregulation in Amoeba proteus

We describe the effects of arginine-vasopressin (AVP) and five related peptides on the contractile vacuole, the osmoregulatory organelle of the fresh water Amoeba proteus. Arginine-vasopressin, lysine-vasopressin, and SKF 101926, a synthetic antagonist of vasopressin, cause a significant increase in the rate of output of the contractile vacuole. Deamino-vasopressin (dAVP), oxytocin, and arginine-vasotocin have no such activity, although dAVP interferes with the action of AVP when present in equimolar concentration. Relatively high concentrations are required and the effect of active peptides is readily reversible. When the normal, hypotonic medium (a synthetic pond water) is replaced by isotonic sucrose, the action of AVP on the vacuole is abolished. Thus vasopressin is believed to act by increasing permeability of the Amoeba plasma membrane to water.