The efficiency of different IRESs (internal ribosomes entry site) in monocistronic mRNAS.

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 stimulation of gene expression by the R region from HTLV-1 and BLV.

The 5' untranslated regions (5'UTR) of mRNA are known to stimulate or inhibit more or less translation. SR alpha, an association of SV40 early gene promoter and of the R region plus the first 39 nucleotides of the U5 region (designated as R) from the human T-cell leukemia virus (HTLV-1) is currently used to stimulate expression of various coding regions. Its effect is considered to take place at the translational level. In all studies published so far, the R region was associated with the promoter and 5'UTR from SV40 early genes. In the present work, the role of SV40 5'UTR and HTLV-1R region was evaluated separately using different promoters, reporter genes and cells. Both SV40 5'UTR (SU) and R region (R) from HTLV-1 stimulated separately the expression of adjacent reporter genes. When associated, the SV40 5'UTR and the R region from HTLV-1 (SUR) were a more potent stimulator of gene expression and their effects were more than additive. This effect was very potent in HeLa and HC11 cells and almost inexistent in CHO and COS 7 cells. It was of various intensity in other cell types including bird and fish cells. The presence of SUR in gene constructs favoured the accumulation of the mRNAs. SUR stimulated gene expression when added between the cap and the initiation codon. Unexpectedly, SUR was never inhibitory. SUR can therefore be considered essentially as potent and specific stimulator of gene expression favoring mRNA accumulation.

The optimal use of IRES (internal ribosome entry site) in expression vectors.

In higher eucaryotes, natural bicistronic mRNA have been rarely found so far. The second cistron of constructed bicistronic mRNAs is generally considered as not translated unless special sequences named internal ribosome entry site (IRES) are added between the two cistrons. These sequences are believed to recruit ribosomes independently of a cap structure. In the present report, a new IRES found in the HTLV-1 genome is described. A systematic study revealed that this IRES, but also the poliovirus (polio) and the encephalomyocarditis virus (EMCV) IRES work optimally when they are added about 100 nucleotides after the termination codon of the first cistron. Unexpectedly, these IRES became totally inefficient when added after 300-500 nucleotide spacers. This result and others are not compatible with the admitted mechanism of IRES action. The IRES appear to be rather potent translation stimulators. Their effects are particularly emphasized in cells in which the normal mechanism of translation initiation is inhibited. For these reasons, we suggest to call IRES rescue translation stimulators (RTS).

Effect of intercistronic length on internal ribosome entry site (IRES) efficiency in bicistronic mRNA.

Specific structures found in the mRNA of picornavirus are known to allow a cap-independent translation. These structures, named internal ribosome entry sites (IRES), are also able to favor translation of the second cistron in bicistronic mRNAs. Their mechanism of action is not well understood. In the present study, two IRESs have been used: the IRES from poliovirus and a newly discovered IRES (SUR) composed of the 5' P untranslated sequence from SV40 early genes, the R structure, and a small part of the U5 region from the human leukemia virus-1 (HTLV-1). The bicistronic constructs containing the firefly luciferase gene as the first cistron and the chloramphenicol acetyltransferase (CAT) as the second cistron were driven by the Rous sarcoma virus (RSV) promoter and contained the early gene SV40 terminator. All the resulting plasmids were tested by transfection in HeLa and CHO cells. In the bicistronic mRNAs without IRES, the expression of the CAT gene was dependent on the distance between the two cistrons. The maximum efficiency in the expression of the second cistron was obtained when the intercalating RNA was composed of 30 to 90 nucleotides. This expression was deeply reduced when the intercalating fragment contained 8 or 300 nucleotides and was undetectable with 500 nucleotides. Unexpectedly, the luciferase mRNA was almost not expressed when the intercalating RNA was of 8 or 30 nucleotides. Expression of the luciferase gene occurred when the intercistronic RNA fragment was of 80 nucleotides and it became lower at 300 and 500 nucleotides. The same observations were done when the poliovirus or the SUR IRESs were added after the intercistronic spacers. However, expression of the CAT gene was amplified by both IRESs. When the CAT cistron preceded by the poliovirus or SUR IRES was introduced within luciferase cistron, 316 nucleotides before its termination codon, the IRESs were able to initiate translation of the following CAT gene irrespectively of the mRNA luciferase reading frame. Moreover, with all these constructs the highest expression level of the CAT cistron did not exceed 10% of that obtained with the same vector carrying only the CAT cistron. To identify a possible relation between the IRESs and the cap site, the CAT cistron preceded or not with an IRES was introduced 210 nucleotides downstream of the AUG codon of the luciferase gene (i.e., 258 nucleotides from the cap site) and 100 nucleotides after an added UAG termination codon. Expression of the CAT gene was not modified by the addition of the poliovirus IRES but it was strongly stimulated by the SUR IRES (the level of expression corresponded to 65% of that obtained with the same vector carrying only the CAT cistron). These results suggest that there is a cooperation between the cap and the SUR IRES and not the poliovirus IRES to stimulate translation. These data indicate that IRESs must be introduced in precise position to allow an efficient expression of the second cistron in bicistronic mRNAs.

A newly established porcine aortic endothelial cell line: characterization and application to the study of human-to-swine graft rejection.

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.

Recombinant expression and secretion of a natural splicing variant containing the ectodomain of porcine LH receptor in HC11 mammary epithelial cells.

Large-scale synthesis of active recombinant porcine luteinizing hormone/chorionic gonadotropin receptor (pLHR) is required for biophysical and structural studies. This study was undertaken to improve expression of the corresponding cDNA already obtained with a number of other systems, (i) by turning to cells from mammalian origin able to perform adequate glycosylation, (ii) by using an expression vector containing the acknowledged high-performance rabbit WAP gene upstream region together with transcription and translation stimulating sequences, and (iii) by expressing natural splicing variants. Selection of the transfected HC11 cells was performed in terms of pLHR expression using specific radioligand binding and immunoradiometric assays. Secretion of pLHR ectodomain into the culture medium of the HC11 clones was quantified, and reached 70 ng/ml, which represents the highest active amount ever produced. However, this level of expression was relatively low in comparison to that currently observed with bGH cDNA used as reporter gene. Additional investigations were performed in order to gain further insight into the limitation of the production of pLHR relative to bovine or human growth hormone using the same expression system. A high number of copies of cDNA in the genome of HC11 cells was found, provided that an antibiotic selection pressure was maintained to avoid drifting. The low mRNA levels detected for pLHR relative to hGH mRNAs correlate well with the relative protein production levels. They could arise from poor stability of mRNAs, a fact already observed for the natural receptor in gonadal cells. These results thus constitute a promising indicator for possible expression of pLHR in the milk of transgenic animals.

The RU5 ('R') region from human leukaemia viruses (HTLV-1) contains an internal ribosome entry site (IRES)-like sequence.

RNA fragments containing the complete R region and the beginning of the U5 region ('R') from the human T cell leukaemia virus 1 (HTLV-1) stimulated the translation of the second cistrons in bicistronic mRNAs. The 5' untranslated region from SV40 early genes (SU) which was unable to stimulate translation of second cistrons amplified markedly the internal ribosome entry site (IRES) effect of the HTLV-1 'R' fragments. The 'R' regions from HTLV-1 have therefore properties similar to internal ribosome entry sites (IRES) originally found in picornavirus. The beginning of the U5 region from HTLV-1 contains a polypyrimidine sequence which is known to play an essential role in the IRES activity in picornavirus. The same experiments carried out using the 'R' region from bovine leukaemia virus (BLV) showed that this sequence has at most a weak IRES effect. One retroviruses, HTLV-1 and perhaps others contain therefore an IRES activity. Interestingly, the combined SU 'R' sequence worked efficiently with different cistrons, different promoters and in all tested cell lines, whereas the poliovirus IRES was active in CHO cells but not in the mouse mammary cell line HC11. The SU 'R' sequence may therefore preferably be used to generate active bicistronic mRNAs.

The deleterious effects of human erythropoietin gene driven by the rabbit whey acidic protein gene promoter in transgenic rabbits.

Human erythropoietin (EPO) gene and cDNA associated with the rabbit whey acidic protein (WAP) gene promoter were used to tentatively produce the recombinant protein in milk of transgenic mice and rabbits. Several gene constructs showed good efficiency in the mouse mammary cell line HC11. None of them was able to direct the expression of the hormone at a concentration higher than 50 micrograms/mL in mouse and rabbit milk. With one of the construct, the rabbits had an abnormally high amount of red blood cells irrespectively of their sex, they could not reproduce and no milk could be obtained from them. These animals died prematurely. In these animals, the EPO gene was therefore expressed at a low but supraphysiological level in organs other than the mammary gland. These experiments show that transgenic animals obtained with gene constructs which do not contain insulators cannot be used as living fermentors to produce human erythropoietin in their milk at an industrial scale.

The effect of various introns and transcription terminators on the efficiency of expression vectors in various cultured cell lines and in the mammary gland of transgenic mice.

Various combinations of promoters, introns and transcription terminators were used to drive the expression of bovine growth hormone (bGH) cDNA in different cell types. In constructs containing the human cytomegalovirus (hCMV) promoter and the SV40 late genes terminator, the intron from SV40 genes (VP1) was much more efficient, than the intron from the early genes (t). The synthetic intron SIS generated by the association of an adenovirus splice donor and an immunoglobulin G splice acceptor showed the highest activity. The respective potency of these introns was similar in several mammalian (CHO, HC11 and COS) and fish (TO2 and EPC) cells. The rabbit whey acidic protein (WAP) gene promoter was highly efficient to drive the expression of bGH gene in the HC11 mammary cell lines. In contrast, the bGH cDNA under the control of the same promoter was much less efficiently expressed when the SV40 VP1 intron and transcription terminator were used. The rabbit WAP gene and the human GH gene terminators did not or only moderately enhanced the expression of the construct WAP bGH cDNA. Introduction of a promoter sequence from the mouse mammary tumor virus (MMTV) LTR in the VP1 intron increased very significantly the expression of the WAP bGH cDNA. Although several of these vectors showed high potency when expressed stably in HC11 cells, all of them were only moderately efficient in transgenic mice. These data indicate that the VP1 and the SIS introns may be used to express foreign cDNAs with good efficiency in different cell types. The addition of an enhancer within an intron may still reinforce its efficiency. However, transfection experiments, even when stable expression is carried out, are poorly predictive of the potential efficiency of a vector in transgenic animals.

The effect of matrix attached regions (MAR) and specialized chromatin structure (SCS) on the expression of gene constructs in cultured cells and in transgenic mice.

The flanking sequences of several genes have been shown to direct a position independent expression of transgenes. Attempts to completely identify the insulating sequences have failed so far. Some of these sequences contain a matrix attached region (MAR) located in the flanking part of the genes. This article will show that the MARs in cultured cells located in the 3' OH region of the human apolipoprotein B100 (Apo B100) and within the SV40 genome were unable to stimulate and insultate transgene expression directed by the promoters from a rabbit whey acidic protein (WAP) gene or from human cytomegalovirus (hCMV) early genes. In transgenic mice, the MAR from the Apo B100 and SV40 genes did not enhance the expression of a transgene containing the rabbit whey acid protein (WAP) promotor, the late gene SV40 intron (VP1 intron), the bovine growth hormone (bGH) cDNA and the SV40 late gene terminator. This construct was even toxic for embryos. Similarly, the specialized chromatin structure (SCS) from the Drosophila 87A7 HSP70 gene reduced chloramphenicol acetyl transferase (CAT) activity when added between a cytomegalovirus (CMV) enhancer and a Herpes simplex thymidine kinase (TK) gene promoter. This inhibitory action was almost complete when a second SCS sequence was added before the CMV enhancer. Sequences from the firefly luciferase and from the human gene cathepsin D cDNA used as control unexpectedly showed a similar inhibitory effect when added to the CMVTKCAT construct instead of SCS. When added before the CMV enhancer and after the transcription terminator in the CMVTKCAT construct, the SCS sequence was unable to insulate the integrated gene as seen by the fact that the level of CAT in cell extracts were by no means correlated with the number of copies in individual clones. From these data, it is concluded that i) a MAR containing the canonical AT rich sequences does not amplify the expression of all gene constructs ii) At rich MAR sequences do not have per se an insulating effect iii) Drosophila SCS from the 87A7 HSP70 gene has no insulating effect in all gene constructs (at least in mammalian cells) iv) and the addition of a DNA fragment between an enhancer and a promoter in a gene construct cannot be used as a reliable test to evaluate its insulating property.

Efficiency of introns from various origins in fish cells.

Several vectors containing (1) regulatory regions from Rous sarcoma virus (RSV), human cytomegalovirus (CMV), and herpes simplex thymidine kinase (TK); (2) introns from early or late SV40 genes and from trout growth hormone gene (tGH); (3) chloramphenicol acetyltransferase gene (CAT); and (4) transcription terminators from SV40 were transfected into carp EPC cells, salmon CHSE cells, tilapia TO2 cells, quail QT6 cells, and hamster CHO cells. CAT activity was measured in extracts from several cell lines 3 days after transfection and in the fish EPC stable clones. The CMV and RSV promoters were the most potent in all cell types. The intron from late SV40 genes (VP1 intron) worked properly in QT6 and CHO cells but not in EPC and very weakly in TO2 cells. The tGH intron was efficient in all cell types but preferentially in fish cells. The small t intron from SV40 was processed in all cell types. The small t and, to a lesser extent, the tGH introns amplified expression of cat gene in stable clones, in comparison to the transiently transfected cells. These results indicate that elements from mammalian genes may not be properly recognized by the fish cellular machinery and in an unpredictable manner. This finding suggests that vectors prepared to express foreign genes in transfected cultured fish cells and transgenic fish should preferably contain DNA sequences from fish genes or, alternatively, those sequences from mammalian genes that have been previously proved to be compatible with the fish cellular machinery.

Gene expression following transfection of fish cells.

Various genes containing different transcriptional regulatory elements (TRE) and the bacterial marker gene coding for chloramphenicol acetyl transferase were transfected into several fish cell lines to evaluate the efficiency of expression in comparison with mammalian cells. The CMV and RSV TRE were the most efficient non-inducible promoters in directing reporter gene expression. RSV and CMV appeared of similar potency in a stable fish cell line. The human HSP-70 promoter showed high potency in a carp and in a trout cell line after thermal induction. This promoter also induced the synthesis of human growth hormone directed by the corresponding cDNA, but not by the gene. RSV TRE was also able to drive the synthesis of bovine growth hormone when attached directly to the cDNA but not to the gene. These data suggest that non-fish gene TRE can be used to express foreign genes in fish cells or transgenic fish; however, in most cases they are relatively inefficient. The data also suggest that the translation and secretion machinery of fish cells can express efficiently foreign genes but that mammalian introns might be not processed properly in some cases.

A distal region enhances the prolactin induced promoter activity of the rabbit alpha s1-casein gene.

Casein gene expression is induced in the rabbit mammary gland by prolactin (PRL). alpha s1-casein is the major casein secreted into milk. In order to define the position of the DNA sequences involved in the control of rabbit alpha s1-casein gene regulation by PRL, chimeric genes were constructed between upstream regions of the rabbit alpha s1-casein gene and the chloramphenicol acetyl transferase (CAT) reporter gene. A series of 5'-deleted fusion genes was obtained by nuclease digestion of the alpha s1-casein gene upstream region. These gene constructs were transfected into rabbit primary mammary cells, or cotransfected in CHO cells with the plasmid coding for the rabbit mammary receptor (PRL-R). A regulatory region has been located between nt -3768 and -3155. This region enhances the prolactin induced promoter activity of the alpha s1-casein gene. It might possess or cooperate with prolactin responsive elements located further downstream in the alpha s1-casein gene.

Ultrastructure of in vivo fertilization in the goat.

In vivo fertilization of goat eggs has been studied by electron microscopy. Eggs were recovered from superovulated or natural cyclic goats, 32 to 52 hours after the onset of oestrus; only eggs recovered between 46 and 52 hours were fertilized. Spermatozoa penetrated the zona pellucida tangentially leaving vesiculated products of the acrosome reaction at the zona surface. As sperm penetrated into the ooplasm, the second meiotic division completed and cortical granule exocytosis occurred. However a few unreacted cortical granules usually remained in the cortex of the fertilized eggs, adjacent to the plasma membrane. After swelling the two pronuclei presented similar ultrastructural morphology: they contained small, compact, agranular nucleoli and unevenly distributed chromatin. The cytoplasm in close vicinity to the apposed pronuclei contained large stacks of annulate lamellae, smooth endoplasmic reticulum, prominent Golgi complexes, as well as dense areas of unidentified material. The abundance of cytoplasmic organelles near the pronuclei might be the expression of intensive metabolic activity. Conversely, in the cortex of fertilized ova several large organelles-free cytoplasmic areas were randomly distributed.

In vitro fertilization with normal development in the sheep.

Ovine tubal (n = 87) and ovarian in vitro matured oocytes (n = 99) were fertilized in vitro with ejaculated spermatozoa capacitated for 8 h in modified defined medium buffered with Hepes. High levels of fertilization were obtained as assessed by development to two- to six-cell stage within 40 h (75.8% for ovulated and 62.6% for in vitro matured oocytes). Electron microscope analysis of oocytes 20-22 h after insemination indicated that in vitro fertilization approximated the in vivo events. Embryos (two- to six-cell) were transferred surgically to the oviducts of pseudopregnant rabbits. Three days later, 42 (from ovulated oocytes) and 15 (from in vitro matured oocytes) embryos were recovered; 26 (61.9%) and 10 (66.6%), respectively, had cleaved at least once. Embryos incubated in vivo (n = 20 from ovulated oocytes; n = 9 from in vitro matured oocytes) were transferred surgically to the uteri of seven and four recipient ewes resulting in four and two pregnancies, respectively, from which three and one, respectively, have been maintained (greater than 3 months). The first lamb resulting from the in vitro fertilization of an ovulated oocyte was born. In addition, six embryos (two- to four-cell) from tubal oocytes and ten embryos (two- to six-cell) from in vitro matured oocytes were directly transferred to the oviducts of two and three ewes, respectively. Two pregnancies resulting from in vitro matured fertilized oocytes are in progress (greater than 3 months).