Production of transgenic birds

The avian embryo presents a tremendous challenge for those interested in accessing and manipulating the avian germ line. By far the most successful method of gene transfer is by retrovirus vector. The efficacy of retrovirus vectors has been demonstrated by germ line insertion of replication-competent retroviruses as well as the insertion of replication-defective retrovirus vectors carrying bacterial marker genes. Retroviral vectors have also been shown to be useful for the transfer and expression of genes in somatic cells. Further, germ line transgenesis has been reported in both the chicken and the Japanese quail. In addition, several alternative gene transfer methods are under development. These include transfection of avian sperm, development of germ line chimeras using primordial germ cells and blastodermal cells, and the development of embryonic stem cell lines. Potentially, basic research and the poultry industry will derive substantial benefit from this revolutionary technology.     

Production of transgenic birds

The avian embryo presents a tremendous challenge for those interested in accessing and manipulating the avian germ line. By far the most successful method of gene transfer is by retrovirus vector. The efficacy of retrovirus vectors has been demonstrated by germ line insertion of replication-competent retroviruses as well as the insertion of replication-defective retrovirus vectors carrying bacterial marker genes. Retroviral vectors have also been shown to be useful for the transfer and expression of genes in somatic cells. Further, germ line transgenesis has been reported in both the chicken and the Japanese quail. In addition, several alternative gene transfer methods are under development. These include transfection of avian sperm, development of germ line chimeras using primordial germ cells and blastodermal cells, and the development of embryonic stem cell lines. Potentially, basic research and the poultry industry will derive substantial benefit from this revolutionary technology.     

SET domain proteins modulate chromatin domains in eu- and heterochromatin

The SET domain is a 130-amino acid, evolutionarily conserved sequence motif present in chromosomal proteins that function in modulating gene activities from yeast to mammals. Initially identified as members of the Polycomb- and trithorax-group (Pc-G and trx-G) gene families, which are required to maintain expression boundaries of homeotic selector (HOM-C) genes, SET domain proteins are also involved in position-effect-variegation (PEV), telomeric and centromeric gene silencing, and possibly in determining chromosome architecture. These observations implicate SET domain proteins as multifunctional chromatin regulators with activities in both eu- and heterochromatin – a role consistent with their modular structure, which combines the SET domain with additional sequence motifs of either a cysteine-rich region/zinc-finger type or the chromo domain. Multiple functions for chromatin regulators are not restricted to the SET protein family, since many trx-G (but only very few Pc-G) genes are also modifiers of PEV. Together, these data establish a model in which the modulation of chromatin domains is mechanistically linked with the regulation of key developmental loci (e.g. HOM-C).     

A quick and efficient method to generate mammalian stable cell lines based on a novel inducible alphavirus DNA/RNA layered system

We report a new method to generate high-expressing mammalian cell lines in a quick and efficient way. For that purpose, we developed a master cell line (MCL) containing an inducible alphavirus vector expressing GFP integrated into the genome. In the MCL, recombinant RNA levels increased >4,600-fold after induction, due to a doxycycline-dependent RNA amplification loop. The MCL maintained inducibility and expression during 50 passages, being more efficient for protein expression than a conventional cell line. To generate new cell lines, mutant LoxP sites were inserted into the MCL, allowing transgene and selection gene exchange by Cre-directed recombination, leading to quick generation of inducible cell lines expressing proteins of therapeutic interest, like human cardiotrophin-1 and oncostatin-M at several mg/l/24 h. These proteins contained posttranslational modifications, showed bioactivity, and were efficiently purified. Remarkably, this system allowed production of toxic proteins, like oncostatin-M, since cells able to express it could be grown to the desired amount before induction. These cell lines were easily adapted to growth in suspension, making this methodology very attractive for therapeutic protein production.     

Chromatin domain boundaries in the Bithorax complex

Eukaryotic chromosomes are thought to be organized into a series of discrete higher-order chromatin domains. This organization is believed to be important not only in the compaction of the chromatin fibre, but also in the utilization of genetic information. Critical to this model are the domain boundaries that delimit and segregate the chromosomes into units of independent gene activity. In Drosophila, such domain boundaries have been identified through two different approaches. On the one hand, elements like scs/scs′ and the reiterated binding site for the SU(HW) protein have been characterized through their activity of impeding enhancer-promoter interactions when intercalated between them. Their role of chromatin insulators can protect transgenes from genomic position effects, thereby establishing in dependent functional domains within the chromosome. On the other hand, domain boundaries of the Bithorax complex (BX-C) like Fab-7 and Mcp have been identified through mutational analysis. Mcp and Fab-7, however, may represent a specific class of boundary elements; instead of separating adjacent domains that contain separate structural genes, Mcp and Fab-7 delimit adjacent cis-regulatory domains, each of which interacts independently with their target promoters. In this article, we review the genetic and molecular characteristics of the domain boundaries of the BX-C. We describe how Fab-7 functions to confine activating as well as repressive signals to the flanking regulatory domains. Although the mechanisms by which Fab-7 works as a domain boundary remain an open issue, we provide preliminary evidence that Fab-7 is not a mere insulator like scs or the reiterated binding site for the SU(HW) protein.     

Selectable genetic markers for nematode transgenesis

The nematode Caenorhabditis elegans has been used to study genetics and development since the mid-1970s. Over the years, the arsenal of techniques employed in this field has grown steadily in parallel with the number of researchers using this model. Since the introduction of C. elegans transgenesis, nearly 20 years ago, this system has been extensively used in areas such as rescue experiments, gene expression studies, and protein localization. The completion of the C. elegans genome sequence paved the way for genome-wide studies requiring higher throughput and improved scalability than provided by traditional genetic markers. The development of antibiotic selection systems for nematode transgenesis addresses these requirements and opens the possibility to apply transgenesis to investigate biological functions in other nematode species for which no genetic markers had been developed to date.     

Thyroid hormone receptor-mediated regulation of the methionine adenosyltransferase 1 gene is associated with cell invasion in hepatoma cell lines

The thyroid hormone T₃ regulates differentiation, growth, and development. We demonstrated that methionine adenosyltransferase 1A (MAT1A) was positively regulated by T₃ identified by cDNA microarray previously. The expression of the MAT1A was upregulated by T₃ in hepatoma cell lines overexpressing thyroid hormone receptors (TRs). Additionally, these findings indicate that MAT1A may be regulated by CCAAT/enhancer binding protein (C/EBP). The critical role of the C/EBP binding sites was confirmed by the reporter or chromatin immuno-precipitation (ChIP) assay. In addition, C/EBP was upregulated in hepatoma cells after T₃ treatment and ectopic expression of MAT1A inhibited cell migration and invasion in J7 hepatoma cells. Conversely, knockdown of MAT1A expression increased cell migration. Together, these findings suggest that the expression of the MAT1A gene is mediated by C/EBP and is indirectly upregulated by T₃. Finally, TR was downregulated in a small subset of hepatocellular carcinoma cells concomitantly reduced the expression of C/EBPα and MAT1A.     

Emerging connections between DNA methylation and histone acetylation

Modifications of both DNA and chromatin can affect gene expression and lead to gene silencing. Evidence of links between DNA methylation and histone hypoacetylation is accumulating. Several proteins that specifically bind to methylated DNA are associated with complexes that include histone deacetylases (HDACs). In addition, DNA methyltransferases of mammals appear to interact with HDACs. Experiments with animal cells have shown that HDACs are responsible for part of the repressive effect of DNA methylation. Evidence was found in Neurospora that protein acetylation can in some cases affect DNA methylation. The available data suggest that the roles of DNA methylation and histone hypoacetylation, and their relationship with each other, can vary, even within an organism. Some open questions in this emerging field that should be answered in the near future are discussed.     

Transgenesis in fish

Gene transfer into fish embryo is being performed in several species (trout, salmon, carps, tilapia, medaka, goldfish, zebrafish, loach, catfish, etc.). In most cases, pronuclei are not visible and microinjection must be done into the cytoplasm of early embryos. Several million copies of the gene are generally injected. In medaka, transgenesis was attempted by injection of the foreign gene into the nucleus of oocyte. Several reports indicate that the injected DNA was rapidly replicated in the early phase of embryo development, regardless of the origin and the sequence of the foreign DNA. The survival of the injected embryos was reasonably good and a large number reached maturity. The proportion of transgenic animals ranged from 1 to 50% or more, according to species and to experimentators. The reasons for this discrepancy have not been elucidated. In all species, the transgenic animals were mosaic. The copy number of the foreign DNA was different in the various tissues of an animal and a proportion lower than 50% of F1 offsprings received the gene from their parents. This suggests that the foreign DNA was integrated into the fish genome at the two cells stage or later. An examination of the integrated DNA in different cell types of an animal revealed that integration occurred mainly during early development. The transgene was found essentially unrearranged in the fish genome of the founders and offsprings. The transgenes were therefore stably transmitted to progeny in a Mendelian fashion. Southern blot analysis revealed the presence of possible junction fragments and also of minor bands which may result from a rearrangement of the injected DNA. In all species, the integrated DNA appeared mainly as random end-to-end concatemers. In adult trout blood cells, a small proportion of the foreign DNA was maintained in the form of non-integrated concatemers, as judged by the existence of end fragments. The transgenes were generally only poorly expressed. The majority of the injected gene constructs contained essentially mammalian or higher vertebrates sequences. The comparison of the expression efficiency of these constructs in transfected fish and mammalian cells indicates that some of the mammalian DNA sequences are most efficiently understood by the fish cell machinery. Chloramphenicol acetyl transferase gene under the control of promoters from Rous sarcoma virus, and human cytomegalovirus, was expressed in several tissues of transgenic fish. Chicken -crystallin gene was expressed in several tissues of transgenic fish. Rainbow trout growth hormone cDNA driven by the Rous sarcoma virus promoter was expressed in transgenic carps leading to a faster growth of these animals. The antifreeze protein gene from flounder was expressed in transgenic salmon. These data indicate that transgenesis in fish is relatively easy but that fish gene sequences must be preferably used to obtain a good expression of the transgenes. Fish is a good biological model, specially for developmental studies and it is an increasing part of human food. For these reasons, transgenesis in fish is most likely to be more and more practised in the coming years.