[The exploration and practice of production of transgenic zebrafish into undergraduate student gene engineering experimental teaching].

The preparation of transgenic animals is one of the core technology and critical achievement of gene engineering. However, it has not been reported that the gene engineering experimental course of undergraduate students in universities of mainland China has carried out the preparation of transgenic animals. In this paper, the authors took the advantage of scientific research platform, introduced the transgenic zebrafish technology to gene engineering experimental course of undergraduate students, and explored and practiced related teaching model, which had achieved good results and had great value to popularize.

Role of svp in Drosophila pericardial cell growth.

The Drosophila dorsal vessel is a segmentally repeated linear organ, in which seven-up (svp) is expressed in two pairs of cardioblasts and two pairs of pericardial cells in each segment. Under the control of hedgehog (hh) signaling from the dorsal ectoderm, svp participates in diversifying cardioblast identities within each segment. In this experiment, the homozygous embryos of svp mutants exhibited an increase in cell size of Eve positive pericardial cells (EPCs) and a disarranged expression pattern, while the cardioblasts pattern of svp-lacZ expression was normal. In the meantime, the DAI muscle founders were absent in some segments in svp mutant embryos, and the dorsal somatic muscle patterning was also severely damaged in the late stage mutant embryos, suggesting that svp is required for the differentiation of Eve-positive pericardial cells and DA1 muscle founders and may have a role in EPC cell growth.

[The use of RNAi as a technique to study the functions of heart-related genes in Drosophila].

RNAi is a recently developed method to block the activity of cellular genes by artificially providing sense and anti-sense RNA corresponding to a target gene. By inducing rapid degradation of the corresponding endogenous mRNA and blocking new mRNA synthesis, RNAi leads to post-transcriptional gene silencing. Now this phenomenon has been claimed to exist in C. elegans, Drosophila, buds, fungi and plants and is being used to study the functions of some special genes or the known genes at specific time point. It is extremely useful for those genes or organisms that their mutants are not easily obtained. The Drosophila heart related genes, tinman and wingless, have been shown to play an important role in coordinating the early formation of heart progenitor cells and precursors, yet the late function is still unexplored. In this experiment, we took the advantage of RNAi technique, microinjected tinman and wingless dsRNA into the early embryos in Drosophila respectively and got these two genes' RNAi phenotypes, which were very similar to that of their mutant, showing heart tube defects or no heart precursors formation. tinman dsRNA even caused visceral mesoderm defects and the somatic muscles disruption, yet wingless dsRNA only affected heart precursors and had no effect on visceral mesoderm and somatic muscles, indication that the heart-related genes dsRNA interference worked effectively and exclusively in Drosophila.

[Functional analysis of the human T-type calcium channel alpha 1H subunit gene in cellular proliferation].

An increase in the functional expression of the T-type calcium (Ca) channel previously has been proposed to be associated with vascular smooth muscle cell proliferation although direct evidence for the channel causing these effects remains to be demonstrated. In this study, we provide evidence that stable over-expression of the alpha 1H subunit of the T-type Ca channel (CACNA1H) in HEK-293 cells confers a significant growth advantage. Over-expression of the alpha 1H subunit of the T-channel was confirmed in stable transfects by RT-PCR analysis using specific primer pairs and also by electrophysiology. Growth curve assays showed population doubling time for alpha 1H stable transfects was 14.0 +/- 0.4 h, whereas control cultures had a population doubling time of 22.1 +/- 1.1 h (n = 3, P < 0.05). In addition, total cell numbers significantly increased in stable transfects at all time points investigated from 48-120 h after plating (5 x 10(3) cells/well) compared with control cultures. Consistent with these findings flow cytometry showed that 53.9% of control cells were in G1, 34.5% in S and 11.6% in G2/M whereas alpha 1H transfectants displayed 45.6% of cells in G1, 44.6% in S and 9.8% in G2/M. Finally, the Western blotting results verified that the levels of protein expression of CDK2, cyclin A and cyclin E were relatively high in alpha 1H transfectants compared to control cultures. Our results demonstrate that the T-type Ca channel provides a growth advantage to HEK-293 cells when stably expressed and that it probably exerts these effects via control of the G1/S cell cycle mechanism.

[Establishment and application of double color and fiber-FISH].

To determine whether genetic material in 5q13.3 breakpoint region changed in the course of establishing the cell line with a constitutional chromosomal inversion inv(5)(p13.1q13.3) associated with hairy cell leukemia, double color fluorescence in situ hybridization (FISH) on metaphase, interphase and DNA fibers were performed in cells of the cell line with the cosmids cCI5-216 and cCI5-267 DNA probes labeled by either biotinylate or digoxigenin. It showed that the cell line gave the same results as those of the original cells, which indicated that no change of the genetic material at 5q13.3 breakpoint region occurred. The cell line is valuable to reveal the molecular pathogenesis of hairy cell leukemia.

[Functional analysis of the alpha1G subunit of the T-type calcium channel in cellular proliferation].

Increases of functional T-type calcium channel (T-channel) expression have been associated with cellular proliferation although evidence for this remains controversial. In the present study, we have used a variety of cellular, molecular and electrophysiological techniques to test the hypothesis that T-type channels play a causal role in the signaling pathway leading to proliferation. The results showed that stable over-expression of alpha1G T-channel subunit in HEK-293 cells conferred a significant growth advantage. Thus, cell population doubling time was reduced to 13.7 +/- 0.3 h in alpha1G transfectants, compared to control cultures (22.1 +/- 1.1 h) and flow cytometry analysis showed that this was due to a reduction in the number of alpha1G transfectants residing in the G0/G1 phases of the cell cycle compared to controls. The selective T-type calcium channel blocker, mibefradil, induced a dose-dependent inhibition of proliferation in alpha1G tansfectants. Furthermore, the Western blotting results proved that the level of protein expression of CDK2, cyclin A and cyclin E was high in alpha1G transfectants compared to control cultures. Our results demonstrate that the T-type calcium channel provides a significant growth advantage to HEK-293 cells that might occur via effects on the G1/S cell cycle mechanism.

[Inhibition of vascular smooth muscle cell proliferation by non-steroidal anti-inflammatory drugs].

Abnormal vascular smooth muscle cell (VSMC) proliferation is known to play an important role in the pathogenesis of atherosclerosis, restenosis and instent stenosis. Recent studies suggest that salicylates, in addition to inhibiting cyclooxygenase activity, exert an antiproliferative effect on VSMC growth both in vitro and in vivo. However, whether all non-steroidal anti-inflammatory drugs (NSAID) exert similar antiproliferative effects on VSMCs, and do so via a common mechanism of action, remains unknown. In the present study, we demonstrated that the NSAIDs, aspirin, ibuprofen and sulindac induced a dose-dependent inhibition of proliferation in rat A10 VSMCs (IC50 = 1666 mumol/L, 937 mumol/L and 520 mumol/L, respectively). These drugs did not show significant cytotoxic effects as determined by LDH release assay, even at the highest concentrations tested (aspirin, 5000 mumol/L; ibuprofen, 2500 mumol/L; and sulindac, 1000 mumol/L). Flow cytometric analyses showed that a 48 h exposure of A10 VSMCs to ibuprofen (1000 mumol/L) and sulindac (750 mumol/L) led to a significant G1 arrest (from 68.7 +/- 2.0% of cells in G1 to 76.6 +/- 2.2% and 75.8 +/- 2.2%, respectively, p < 0.05). In contrast, aspirin (2500 mumol/L) failed to induce a significant G1 arrest (68.1 +/- 5.2%). Clearer evidence of a G1 block was obtained by treatment of cells with the mitotic inhibitor, nocodazole (40 ng/ml), for the final 24 h of the experiment. Under these conditions, aspirin still failed to induce a G1 arrest (from 25.9 +/- 10.9% of cells in G1 to 19.6 +/- 2.3%) whereas ibuprofen and sulindac led to a significant accumulation of cells in G1(51.8% +/- 17.2% and 54.1% +/- 10.6%, respectively, p < 0.05). These results indicate that ibuprofen and sulindac inhibit VSMC proliferation by arresting the cell cycle in the G1 phase whereas the effect of aspirin appears to be independent of any special phase of the cell cycle. Irrespective of mechanism, our results suggest that NSAIDs might be of benefit to the treatment of vascular proliferative disorders.