Development of a Mucus Gland Bioreactor in Loach Paramisgurnus dabryanus

Most currently available bioreactors have some defects in the expression, activity, or purification of target protein and peptide molecules, whereas the mucus gland of fish can overcome these defects to become a novel bioreactor for the biopharmaceutical industry. In this study, we have evaluated the practicability of developing a mucus gland bioreactor in loach (Paramisgurnus dabryanus). A transgenic construct pT2-krt8-IFN1 was obtained by subcloning the promoter of zebrafish keratin 8 gene and the type I interferon (IFN1) cDNA of grass carp into the SB transposon. The IFN1 expressed in CIK cells exhibited an antiviral activity against the replication of GCRV873 and activated two genes downstream of JAK-STAT signaling pathway. A transgenic loach line was then generated by microinjection of the pT2-krt8-IFN1 plasmids and in vitro synthesized capped SB11 mRNA. Southern blots indicated that a single copy of IFN1 gene was stably integrated into the genome of transgenic loach. The expression of grass carp IFN1 in transgenic loaches was detected with RT-PCR and Western blots. About 0.0825 µg of grass carp IFN1 was detected in 20 µL mucus from transgenic loaches. At a viral titer of 1 × 103 PFU/mL, plaque numbers on plates containing mucus from transgenic loaches reduced by 18% in comparison with those of the control, indicating that mucus of IFN1-transgenic loaches exhibited an antiviral activity. Thus, we have successfully created a mucus gland bioreactor that has great potential for the production of various proteins and peptides.

Structural and Functional Dissection of the 5′ Region of the Notch Gene in Drosophila melanogaster

Notch is a key factor of a signaling cascade which regulates cell differentiation in all multicellular organisms. Numerous investigations have been directed mainly at studying the mechanism of Notch protein action; however, very little is known about the regulation of activity of the gene itself. Here, we provide the results of targeted 5′-end editing of the Drosophila Notch gene in its native environment and genetic and cytological effects of these changes. Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) system in combination with homologous recombination, we obtained a founder fly stock in which a 4-kb fragment, including the 5′ nontranscribed region, the first exon, and a part of the first intron of Notch, was replaced by an attachment Phage (attP) site. Then, fly lines carrying a set of six deletions within the 5′untranscribed region of the gene were obtained by ΦC31-mediated integration of transgenic constructs. Part of these deletions does not affect gene activity, but their combinations with transgenic construct in the first intron of the gene cause defects in the Notch target tissues. At the polytene chromosome level we defined a DNA segment (~250 bp) in the Notch5′-nontranscribed region which when deleted leads to disappearance of the 3C6/C7 interband and elimination of CTC-Factor (CTCF) and Chromator (CHRIZ) insulator proteins in this region.

RNAi-Mediated Knockdown of IKK1 in Transgenic Mice Using a Transgenic Construct Containing the Human H1 Promoter

Inhibition of gene expression through siRNAs is a tool increasingly used for the study of gene function in model systems, including transgenic mice. To achieve perdurable effects, the stable expression of siRNAs by an integrated transgenic construct is necessary. For transgenic siRNA expression, promoters transcribed by either RNApol II or III (such as U6 or H1 promoters) can be used. Relatively large amounts of small RNAs synthesis are achieved when using RNApol III promoters, which can be advantageous in knockdown experiments. To study the feasibility of H1 promoter-driven RNAi-expressing constructs for protein knockdown in transgenic mice, we chose IKK1 as the target gene. Our results indicate that constructs containing the H1 promoter are sensitive to the presence of prokaryotic sequences and to transgene position effects, similar to RNApol II promoters-driven constructs. We observed variable expression levels of transgenic siRNA among different tissues and animals and a reduction of up to 80% in IKK1 expression. Furthermore, IKK1 knockdown led to hair follicle alterations. In summary, we show that constructs directed by the H1 promoter can be used for knockdown of genes of interest in different organs and for the generation of animal models complementary to knockout and overexpression models.

Ultrastructural Pathology of Neurofibrillary Tangles in Transgenic Mice Carrying Mutant (P301l) Human Tau Gene

Tau is a microtubule-associated protein that promotes polymerization and stabilization of microtubules. It is the major component of fibrillary neuronal and glial inclusions in Alzheimer's disease (AD) and the tauopathies. An autosomal dominant familial tauopathy, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), has been shown to be caused by pathogenic tau mutations. Neuropathologic features of FTDP-17 are filamentous tau aggregates in neurons and glia. Previous transgenic mice carrying the normal human tau gene developed motor neuron disease and had dystrophic axons, but no neurofibrillary tangles (NFT). In this report we describe ultrastructural features of NFT in transgenic mice carrying a mutant human tau gene. The mice were generated with a tau cDNA containing exon 1, 4, 5, 7, 9, 10, 11-13, intron 13 and exon 14, driven by the mouse prion promoter (MoPrP) and containing the most common FTDP-17 mutation, P301L. The transgenic construct was generated by ligating the P301L tau cDNA with the mouse prion promoter.

A globin enhancer acts by increasing the proportion of erythrocytes expressing a linked transgene.

Enhancer elements have been shown to affect the probability of a gene establishing an active transcriptional state and suppress the silencing of reporter genes in cell lines, but their effect in transgenic mice has been obscured by the use of assays that do not assess expression on a cell-by-cell basis. We have examined the effect of a globin enhancer on the variegation of lacZ expression in erythrocytes of transgenic mice. Mice carrying lacZ driven by the alpha-globin promoter exhibit beta-galactosidase (beta-Gal) expression in only a very small proportion of embryonic erythrocytes. When the transgenic construct also contains the (alphaHS-40 enhancer, which controls expression of the alpha-globin gene, expression is seen in a high proportion of embryonic erythrocytes, although there are variations between transgenic lines which can be attributed to different sites of integration. Analysis of beta-Gal expression levels suggests that expressing cells in lines carrying only the alpha-globin promoter express as much beta-Gal as those in which the transgene also contains alphaHS-40. A marked decline in transgene expression occurs as mice age, which is mainly due to a decrease in the proportion of cells expressing the transgene. Thus, a globin enhancer can act to suppress variegation of a linked transgene; this result is consistent with a model in which enhancers act to establish and maintain an active domain without directly affecting the transcriptional rate.