Understanding the Role of Dicer in Astrocyte Development

The Dicer1 allele is used to show that microRNAs (miRNAs) play important roles in astrocyte development and functions. While it is known that astrocytes that lack miRNAs are dysregulated, the in vivo phenotypes of these astrocytes are not well understood. In this study, we use Aldh1l1-EGFP transgene, a marker of astrocytes, to characterize mouse models with conditional Dicer1 ablation (via either human or mouse GFAP-Cre). This transgene revealed novel features of the defective astrocytes from the absence of miRNA. Although astrocyte miRNAs were depleted in both lines, we found histological and molecular differences in the Aldh1l1-EGFP cells between the two Cre lines. Aldh1l1-EGFP cells from hGFAP-Cre mutant lines displayed up-regulation of Aldh1l1-EGFP with increased proliferation and a genomic profile that acquired many features of wildtype primary astrocyte cultures. In the young mGFAP-Cre mutant lines we found that Aldh1l1-EGFP cells were disorganized and hyperproliferative in the developing cerebellum. Using the Aldh1l1-EGFP transgene, our work provides new insights into the roles of miRNAs in astrocyte development and the features of astrocytes in these two mouse models.     

Stable lines of transgenic zebrafish exhibit reproducible patterns of transgene expression

To study the frequency of germ-line transformation and to examine the reproducibility of tissue-specific transgene expression, we produced several lines of transgenic zebrafish expressing a recombinant chloramphenicol acetyltransferase (CAT) gene. Supercoiled plasmids containing both Rous sarcoma virus and SV-40 promoter sequences upstream of the CAT coding region were injected into zebrafish embryos prior to first cleavage. CAT activity could be detected in batches of injected embryos as early as 8 h and up to at least 12 days post-fertilization. Approximately 18% of injected fish raised to maturity exhibited CAT activity in their fins, and approximately 5% of injected fish became stable germ-line transformants. Breeding studies indicated that although transgenic founder fish were frequently germ-line mosaics, transgenic individuals of subsequent generations were fully hemizygous for the transgene marker. The transgenes present in the F1 progeny of four independent lines were relatively well expressed in fin and skin, while lower levels of expression were observed in heart, gill and muscle. Little or no CAT expression was observed in the brain, liver and gonad. A monoclonal antibody directed against the CAT gene product consistently revealed variegated patterns of CAT expression in ectodermally derived fin epidermal cells in three of these lines. These results show that it is possible to efficiently produce stable germ-line transformants of the zebrafish and to observe reproducible tissue-specific patterns of transgene expression in this organism. Possible mechanisms for the variegated expression observed within tissues are also considered.     

利用ES细胞建立可诱导的白血病模型

胚胎干细胞（embryonic stem cells,ESCs）是从囊胚的内细胞团分离出来的多潜能干细胞,具有多向分化的能力。将外源基因导入ES细胞建立转基因动物,对于研究外源基因的功能和调控具有一定的价值。载有外源性基因的病毒在感染ES细胞后,可通过囊胚注射获得具有胚系遗传的该转基因动物,并且这一外源基因可以稳定遗传和表达。该研究主要是利用携带hPML-RARα基因的慢病毒感染小鼠ES细胞系（R1）,获得携带该基因的ES细胞,感染后的ES细胞核型正常。在此基础上,将感染后的ES细胞经囊胚注射,获得了携带有hPML-RARα基因的3只嵌合小鼠,其中,有1只具有遗传特性。对嵌合体小鼠与C57杂交的后代给予强力霉素（doxycycline）处理,3天以后骨髓细胞hPML-RARα基因开始表达,这证明了在小鼠体内该外源基因表达的可诱导性。以上证实,已经成功利用ES细胞建立了可诱导的白血病转基因小鼠模型。     

How to make tetracycline-regulated transgene expression go on and off

Tetracycline-regulated gene expression systems are widely used to allow temporal and quantitative control of transgene expression in cultured cells and transgenic animals. While working with the Tet-Off system, where tetracycline or the analogue doxycycline suppresses expression, we noted a considerable variability in induced transgene expression after removal of doxycycline. Variable expression of the transgene could not be explained by clonal variation since it was noted when working with clonal cell lines. Instead we found that doxycycline bound nonspecifically to cells and extracellular matrix and was slowly released after it had been removed from tissue culture media. The released doxycycline reached sufficiently high levels to completely suppress transgene expression. The effect was not dependent on cell type or the nature of the transgene. However, robust and rapid transgene expression could be induced if released doxycycline were removed by washing cells 3h after the initial removal of doxycycline. The use of different vector systems, harboring the tetracycline-regulatable components, yielded similar results. These results not only help explain why tetracycline-regulatable transgene expression systems sometimes are variable but also provide simple ways to substantially improve the efficiency, utility, and reliability of these widely used expression systems.     

Efficient transgenesis in farm animals by lentiviral vectors

Microinjection of DNA is now the most widespread method for generating transgenic animals, but transgenesis rates achieved this way in higher mammals are extremely low. To address this longstanding problem, we used lentiviral vectors carrying a ubiquitously active promoter (phosphoglycerate kinase, LV-PGK) to deliver transgenes to porcine embryos. Of the 46 piglets born, 32 (70%) carried the transgene DNA and 30 (94%) of these pigs expressed the transgene (green fluorescent protein, GFP). Direct fluorescence imaging and immunohistochemistry showed that GFP was expressed in all tissues of LV-PGK transgenic pigs, including germ cells. Importantly, the transgene was transmitted through the germ-line. Tissue-specific transgene expression was achieved by infecting porcine embryos with lentiviral vectors containing the human keratin K14 promoter (LV-K14). LV-K14 transgenic animals expressed GFP specifically in basal keratinocytes of the skin. Finally, infection of bovine oocytes after and before in vitro fertilization with LV-PGK resulted in transgene expression in 45% and 92% of the infected embryos, respectively.     

Intrachromosomal recombination between attP regions as a tool to remove selectable marker genes from tobacco transgenes

Recombinant genes conferring resistance to antibiotics or herbicides are widely used as selectable markers in plant transformation. Once transgenic material has been selected, the marker gene is dispensable. We report a novel strategy to remove undesirable parts of a transgene after integration into the tobacco genome. This approach is based on the transfer of a vector containing a NPTII gene flanked by two 352 bp attachment P (attP) regions of bacteriophage lambda, and the identification of somatic tissue with deletion events following intrachromosomal recombination between the attP regions. This system was used to delete a 5.9 kb region from a recombinant vector that had been inserted into two different genomic regions. As the attP system does not require the expression of helper proteins to induce deletion events, or a genetic segregation step to remove recombinase genes, it should provide a useful tool to remove undesirable transgene regions, especially in vegetatively propagated species.     

Position effects in mice carrying a lacZ transgene in cis with the beta-globin LCR can be explained by a graded model.

We studied transgenic mice carrying the lacZ reporter gene linked to the erythroid-specific beta-globin promoter and beta-globin locus control region (LCR). Previously, we had demonstrated that the total level of expression of beta-galactosidase enzyme, which is the product of the lacZ gene, varies widely between different transgenic mice due to position effects at the sites of transgene integration. Here, using the X-gal based in situ assay for beta-galactosidase activity, we found that the percent erythroid cells that expressed the transgene also varied widely between the mice. Moreover, a kinetic analysis showed that the average beta-galactosidase content per expressing cell varied both between samples of different transgenic descent and between erythroid cells within each sample, demonstrating that the variable expression of this lacZ transgene was being controlled in a graded manner. These results suggest that the beta-globin LCR enhancers function through a graded model, which is described, rather than the binary mechanism that has been proposed previously for other enhancers.     

Nestin-Cre Mice Are Affected by Hypopituitarism,Which Is Not Due to Significant Activity of the Transgene in the Pituitary Gland

Nestin-Cre mice express Cre recombinase under control of the rat nestin promoter and central nervous system (CNS) enhancer. While endogenous Nestin is expressed in some other tissues including the pituitary gland, Nestin-Cre mice induce recombination predominantly in the CNS. For this reason, they have been widely used to explore gene function or cell fate in the latter. Pituitary hormonal deficiencies, or hypopituitarism, are associated with a wide range of symptoms and with a significant morbidity. These can have a neural and/or a pituitary origin as the gland''s secretions are controlled by the hypothalamus. We report here that Nestin-Cre mice themselves are affected by mild hypopituitarism. Hence, physiological consequences are expected, especially in combination with defects resulting from Cre mediated deletion of any gene under investigation. To further investigate the origin of this phenotype, we re-examined the activity of the transgene. We compared it with expression of Nestin itself in the context of the hypothalamo-pituitary axis, especially in the light of a recent report showing pituitary Nestin-Cre activity, which contrasts with previous data. Our results disagree with those of this recent study and do not support the claim that Nestin positive cells are present in the pituitary anlagen, the Rathke''s pouch (RP). Moreover we did not observe any significant activity in the post-natal pituitary, in agreement with the initial report.     

Recurrent Selection for Transgene Activity Levels in Maize Results in Proxy Selection for a Native Gene with the Same Promoter

High activity levels of a transgene can be very useful, making a transgene easier to evaluate for safety and efficacy. High activity levels can also increase the economic benefit of the production of high value proteins in transgenic plants. The goal of this research is to determine if recurrent selection for activity of a transgene will result in higher activity, and if selection for activity of a transgene controlled by a native promoter will also increase protein levels of the native gene with the same promoter. To accomplish this goal we used transgenic maize containing a construct encoding green fluorescent protein controlled by the promoter for the maize endosperm-specific 27kDa gamma zein seed storage protein. We carried out recurrent selection for fluorescence intensity in two breeding populations. After three generations of selection, both selected populations were significantly more fluorescent and had significantly higher levels of 27kDa gamma zein than the unselected control populations. These higher levels of the 27kDa gamma zein occurred independently of the presence of the transgene. The results show that recurrent selection can be used to increase activity of a transgene and that selection for a transgene controlled by a native promoter can increase protein levels of the native gene with the same promoter via proxy selection. Moreover, the increase in native gene protein level is maintained in the absence of the transgene, demonstrating that proxy selection can be used to produce non-transgenic plants with desired changes in gene expression.     

Recombination technologies for enhanced transgene stability in bioengineered insects

Transposon-based vectors currently provide the most suitable gene transfer systems for insect germ-line transformation and are used for molecular improvement of the Sterile Insect Technique. However, the long time stability of genome-integrated transposon constructs depends on the absence of transposase activity that could remobilize the transposon-embedded transgenes. To achieve transgene stability transposon vectors are usually non-autonomous, lacking a functional transposase gene, and chosen so that endogenous or related transposon activities are not present in the host. Nevertheless, the non-autonomous transposon-embedded transgenes could become unstable by the unintended presence of a mobilizing transposase that may have been undetected or subsequently entered the host species by horizontal gene transfer. Since the field release of transgenic insects will present environmental concerns relating to large populations and high mobility, it will be important to ensure that transgene constructs are stably integrated for maintaining strain integrity and eliminating the possibility for unintentional transfer into the genome of another organism. Here we review efficient methods to delete or rearrange terminal repeat sequences of transposons necessary for their mobility, subsequent to their initial genomic integration. These procedures should prevent transposase-mediated remobilization of the transgenes, ensuring their genomic stability.     

Hybridization of testis-derived stem cells with somatic cells and embryonic stem cells in mice

Somatic cell hybridization is widely used to study the control of gene regulation and the stability of differentiated states. In contrast, the application of this method to germ cells has been limited in part because of an inability to culture germ cells. In this study, we produced germ cell hybrids using germ-line stem (GS) cells and multipotent germ-line stem (mGS) cells. While GS cells are enriched for spermatogonial stem cell (SSC) activity, mGS cells are similar to embryonic stem (ES) cells and originally derived from GS cells. Hybrids were successfully obtained between GS cells and ES cells, between GS cells and mGS cells, and between mGS cells and thymocytes. All exhibited ES cell markers and a behavior similar to ES cells, formed teratomas, and differentiated into somatic cell tissues. However, none of the hybrid cells were able to reconstitute spermatogenesis after microinjection into seminiferous tubules. Analyses of the DNA methylation patterns of imprinted genes also showed that mGS cells do not possess a DNA demethylation ability, which was found in embryonic germ cells derived from primordial germ cells. However, mGS cells reactivated the X chromosome and induced Pou5f1 expression in female thymocytes in a manner similar to ES cells. These data show that mGS cells possess ES-like reprogramming potential, which predominates over-SSC activity.     

Stimulatory and inhibitory action of cytokines on the regulation of hCMV-IE promoter activity in human endothelial cells

Viral promoters are commonly used as regulatory elements in gene therapy vectors due to their strong activity in various cell lines in vitro. However, transgene expression under the control of viral promoters in vivo has been shown to be limited to a short period of time. Several mechanisms for the transient expression of the delivered transgene may be important including deletion of transduced cells or promoter downregulation. Recently we reported that cytokines may either decrease or increase the activity of the human cytomegalovirus (hCMV) promoter in monocytes depending on the differentiation status of the transduced cells. For many applications, the gene of interest has to be delivered into an inflammatory milieu (tumour, ischaemia/reperfusion, vector-induced inflammation etc.). In this report we investigated the influence of various inflammatory cytokines on the hCMV-IE promoter activity in transduced human primary endothelial cells (Huvec) in vitro, which may be the first target cells after gene transfer into different organs. Cultured cells were infected with an E1-deleted adenoviral vector encoding for E. colibeta-galactosidase (Adbeta-gal) driven by the hCMV-IE promoter and incubated either with or without various cytokines. Our results indicate that interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) downregulate promoter activity in endothelial cells whereas, in contrast, tumour necrosis factor (TNF-alpha), interleukin 1beta (IL-1beta) and interleukin 4 (IL-4) increased the promoter activity. These results suggest that inflammatory processes influence the in vivo expression of transferred viral promoter controlled genes of interest.     

Stable Long Term Germ-Line Transmission of Transgene Integration Sites in Mice

Transgenic mice provide a valuable tool in all fields of basic and applied biological and medical research. In this study, we describe the fate of integrated transgenes in the mammalian host genome over a large number of generations. The stability of the germ-line transmission of integrated tyrosinase transgene copies was monitored up to generation F20 in a large number of individuals from seven transgenic mouse lines. Phenotypic and molecular genetic analysis of the offspring both within the different lines and in cross-breeding experiments revealed the high stability of the transgene integration sites in mice. Only very few individuals were affected by a transgene copy loss. These results indicate that, once homozygous transgenic lines are established, breeding programs can be continued to a high number of generations without further stringent molecular genetic analysis.