Transgenic Loach Research Articles

Human TAF-Iα promotes oncogenic transformation via enhancement of cell proliferation and suppression of apoptosis.

Template activating factor-I (TAF-I) is a multifunctional protein involved in various biological processes including the inhibition of histone acetylation, DNA replication, cell cycle regulation, and oncogenesis. Two main TAF-I isoforms with different N-termini, TAF-Iα and TAF-Iβ (SET), are expressed in cells. There are numerous data about functional properties of TAF-Iβ, whereas the effects of TAF-Iα remain largely unexplored. Here, we employed focus formation and cell proliferation assays, TUNEL staining, cytological analysis, and RT-qPCR to compare the effects of human TAF-Iα and TAF-Iβ genes, transiently expressed in Rat2 cells and in Misgurnus fossilis loaches. We found that both TAF-I isoforms possessed equal oncogenic potential in these systems. Furthermore, an overexpression of human TAF-Iα and TAF-Iβ in Rat2 cells promoted their proliferation. Accordingly, the mitotic index was increased in the transgenic loaches expressing human TAF-Iα or TAF-Iβ. TUNEL assay as well as downregulation of p53 gene and upregulation of bcl-2 gene in these transgenic loaches demonstrated that both isoforms suppressed apoptosis. Thus, TAF-Iα isoform exerts the same oncogenic potential as TAF-Iβ, likely by suppressing the apoptosis and promoting cell proliferation.

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.

Expression of the human TRIM14 and its mutant form (P207L) promotes apoptosis in transgenic loaches.

The tripartite-motif (TRIM)14 protein, one of the TRIM family members, was shown to participate in the antiviral and antibacterial defence. Besides, it appears to play an essential role in the processes of oncogenesis. In some types of human tumour cells, TRIM14 has been shown to inhibit apoptosis, while in others-the overexpression of TRIM14 promotes apoptosis. However, whether TRIM14 mediates apoptosis in the normal cells remains unknown. In the present study, we investigated the possible participation of the human TRIM14 gene and its mutant form (620C > T) in the induction of apoptosis in the transgenic larvae loach Misgurnus fossilis L. We observed that the expression of both forms of TRIM14 gene was accompanied by the increase of the frequency of pyknotic nuclei in fish embryos compared to control groups. Accordingly, using the TUNEL assay, the enhanced apoptosis was revealed upon expression of both forms of TRIM14 gene. The transcription of proapoptotic genes (bax, tp53, and casp9) was significantly increased in transgenic loaches expressing human wild-type TRIM14, but remained unchanged upon expression of its mutant form. In addition, the transcription of c-myc was upregulated in transgenic loaches expressing both forms. Thus, it can be assumed that during embryonic development TRIM14 has a proapoptotic effect on the cells via the activation of c-myc, tp53, and bax genes. Apparently, the mutant TRIM14 directs apoptosis via c-myc by p53-independent mechanism.

Alterations in Synthesis and Repair of DNA during the Development of Loach Misgurnus fossilis.

Using a modified radiolabeled primer extension method (we named this modification misGvA—“misincorporation of G versus A”) we have investigated the DNA synthesis and repair at early and late stages of development of loach Misgurnus fossilis. The misincorporation activity of DNA polymerase iota (Pol ι) in wild-type loach could not be detected by this method at any stage of loach development. In transgenic loach overexpressing human Pol ι we have shown that the bypassing of DNA synthesis arrest after incorporation of mismatched nucleotide by Pol ι (the T-stop) was not associated with this enzyme. Non-transgenic loach larvae are virtually lacking the capacity for error correction of DNA duplex containing a mismatched nucleotide. Such repair activity develops only in the adult fish. It appears that the initial stages of development are characterized by more intensive DNA synthesis, while in terminal stages the repair activities become more prominent. The misGvA approach clearly indicates substantial changes in the DNA synthesis intensity, although the role of particular replicative and repair DNA polymerases in this process requires further study.

Growth Performance of Transgenic Mud Loach Misgurnus mizolepis Carrying a GH Transgene Driven by Mud Loach C-Type Lectin Regulator

Growth hormone (GH) transgenesis in fish has the potential to improve aquaculture efficiency and capacity. However, many fast-growing transgenic fish have experienced side effects caused by excess GH expression. To overcome this unwanted issue associated with several GH transgenic mud loach Misgurnus mizolepis lines carrying GH construct driven by a strong <TEX>${\beta}$</TEX>-actin regulator (<TEX>$pml{\beta}$</TEX>-actGH), we performed an alternative version of GH autotransgenesis using a weaker but more stable regulator, the mud loach lectin promoter. GH transgenesis with a pmlectGH construct consisting of the mud loach GH gene driven by the 2.3-kb lectin promoter exhibited significant growth stimulation. However, the extent of the growth acceleration in pmlectGH transgenics (six times maximum when assessed 2 months post hatching) was much less than that in transgenic individuals carrying the <TEX>$pml{\beta}$</TEX>-actGH construct. Additionally, the extraordinary gigantism that was common in <TEX>$pml{\beta}$</TEX>-actGH-transgenic mud loaches was diminished in transgenic loaches harboring the pmlectGH construct. Transgenic founders (pmlectGH) successfully transmitted their transgene into the next generation with up to 41% frequency. Growth stimulation also persisted in the transgenic F1 strains, with a seven-fold increase in maximum body weight at 6 months of age.

Evaluating the Effects of Different Forms of Transgene on Integration Efficiency in the Loach (&lt;i&gt;Misgurnus anguillicaudatus&lt;/i&gt;) Genome

To assess the effects of different transgene forms on integration efficiency, plasmids containing human growth hormone gene under the control of the metallothionein promoter (pMThGH) in circular, linearized forms and lyposome trapped linearized plasmid were introduced into fertilized loach eggs. Prior to microinjection, plasmids were identified for the presence of human growth hormone (hGH) gene by digestion with restriction enzymes (EcoRI, BamHI, BglII) and by polymerase chain reaction (PCR). The linearized form of pMThGH was generated by digesting circular plasmids with BamHI. Liposome trapped linearized plasmid was prepared in the presence of dioleyl phosphatidyl choline, cholesterol and pMThGH. The presence of hGH genes in the genomic DNA of loach developed from microinjected eggs was identified by a PCR method using two hGH-specific primers. PCR products of the positive individuals showed one band corresponding to 479 bp, while those of negative and control individuals showed no corresponding band. Integration rates of hGH genes into fish chromosomes were 6%, 12% and 11% for circular, linearized plasmids and lyposome trapped linearized plasmid, respectively. In accordance with above order, the average body weight of transgenic loach at 6 months was increased by 1.63, 1.73 and 1.68 times over controls, respectively. The results indicate that plasmids in linearized form and lyposome trapped linearized plasmid were more efficiently integrated into loach chromosomes than circular ones, but there was little significant difference in body weight among the 3 groups of transgenic fish.

Time course of foreign gene integration and expression in transgenic fish embryos

Using a nuclear transplantation approach, the integration and expression of the green fluorescent protein (GFP) gene in the embryogenesis of transgenic loach (Misgurnus anguillicaudatus Cantor) have been studied. TheGFP gene expression is first observed at the gastrula stage, which is consistent with the initiation of cell differentiation of fish embryos. The time course of the foreign gene expression is correlated with the regulatory sequences. The expression efficiency also depends on the gene configuration: the expression of pre-integrating circular plasmid at early embryos is higher than that of the linear plasmid. The integration of theGFP gene is first detected at the blastula stage and lasts for quite a long period. When two types of different plasmids are co-injected into fertilized eggs, the behavior of their integration and expression is not identical.

Electroporation of sperm to introduce foreign DNA into the genome of loach (Misgurnus anguillicaudatus)

An antifreeze protein gene promoter from ocean pout (Macrozoarces americanus) linked to a chinook salmon (Oncorhynchus tshawytscha) growth hormone cDNA (opAFPGHc) was introduced into loach (Misgurnus anguillicaudatus) sperm via electroporation under the following conditions: amplitude, 8 kV; burst time, 1.6 s; number of pulses; 28; pulse time, 120 μs; cycle, 6. Compared with the untreated sperm, the fertilization rate driven by the electroporated sperm was decreased slightly, with no significant effect on hatchability. The success rate of gene transfer was 50%. Genomic Southern blotting demonstrated that DNA from transgenic loach contained positive band(s) with higher (37.5%), the same, or lower molecular size as the transferred opAFPGHc fragment. There was a mosaic distribution of exogenous DNA in a wide variety of tissues analyzed. The average weight gain between the 14th and 161th day of the experimental group was 2.5-fold higher than those of the control and mock-treated groups. The body length distribution of the experimental group was rather widely scattered instead of being normally distributed.