The Establishment Of An Embryonic Cell Line SWAU2-BmE Of Silkworm And Study On RAPD And SSR Thereof

As one pillar of cell engineering, insect cell culture is an important aspect of animal cell culture engineering and also a valuable modern biological technology. To date, it has been applied to various fields in biology, medicine and agriculture extensively and intensively. As silkworm is a model insect, culturing insect cells in vitro, researching the cultural cells theoretically and applying them to industry have been attaching great importance over decades. Although a lot of efforts have been made, the number of existing cell lines of silkworm is still deeply deficient, especially those that have the perspective to be applied into industry. After having drawn the frame of silkworm genome, functional genome researches are in great need of providing numerous cell lines of silkworm as research platform. In order to meet with these requirements in cytobiology, developmental biology and molecular biology, establishment of new baculovirus expression vector systems and study on cell engineering in silkworm and transgenic silkworm, we conducted intensive researches on establishing and studying new cell lines of slikworm.This paper exhaustively described the differentiated and transdifferentiated tissues and cells emerging successively in the primary culture of silkworm embryonic tissues in reversal stage for more than one year, we found that the tissues and cells in different degrees of differentiation displayed the ability to multiplication and the multiplication proved to be the result of mitosis. Later, we separated and filtered the high reproducible cell colonies, established the cell line as SWAU2-BmE and yielded following biological characteristics:In shape, SWAU2-BmE cells are relatively single, short shuttle-like or round, and very small. The cell line belongs to adhesive-type epithelium which can be identified easily. Its spread percent rate is higher than 80%.In preservation, the medium being replaced regularly, SWAU2-BmE cell can be kept for more than 150 d at 4℃ and the period is by far longer than that of the cell lines BraE, BmN, Hi5, Sf9, Sf21. All the above-mentioned cell lines except BmE could be resuscitated after 270 d staying in liquid nitrogen.In growth, SWAU2-BmE cell grew by logarithm in the period of 120h to 192h after inoculation. At 192h, the cell concentration came to the peak. The duplized time for the cell line was 51.8h.The chromosomes of SWAU2-BmE cell line was typical of Lepidoptera insects, namely the chromosomes in metaphase were short pole-like or granule-like and the centromeres were dispersed-type. The ratio of duple cells was high up to 89.76%, thus, it belongs to duple ones. It suggested that the cell line well kept the characteristics of the mitosis and the chromosomes ofsilkworm's embryonic cells. Other than that, abnormal mitosis had not been observed.With regard to the susceptibility of SWAU2-BmE to BmNPV, under same conditions, all the controlled cell lines BmE, BmN, Hi5, Sf9, Sf21 didn't produced nucleo-polyhedrovirus BmNPV, even inoculated for 192 h. While, a few nucleopolyhedrovirus could be seen in some SWAU2-BmE cells at 48h. At 96h, 56.17% of the cells were nucleopolyhedrovirus-positive. At 120 h the infection rate rapidly reached to 86.95% and at 192h it arrived at 96.75%, meanwhile the majority of cells had broken. TCID₅₀ value was 1.581×10⁷ and suggested that SWAU2-BmE cell line was highly sensitive to BmNPV.Both RAPD and SSR DNA fingerprint showed that the genetic distance between SWAU2-BmE cell line and its primary material was very short, the former was 0.1 and the latter was 0.133. These revealed that the cell line well preserved the species' properties of the primary material. The results of clustering analysis to SWAU2-BmE, BmE , BmN , Sf9 , Hi5, Sf21 and the primary material based on these two methods were identical, namely the 7 samples could be clustered into as two classes, one of which consisted of the four species from silkworm and the other consisted of the rest, i.e., two species from Spodoptera frugiperda (Sf9 and Sf21) clustered together, then clustered with Hi5 which came from Trchoplusia ni. It showed the genetic distance between cell lines from same species was smaller than that between cell lines from different species, meanwhile, it also implied that the difference between Bombycidae and Noctuidae genome was obvious. The statistical result that the genetic distance between SWAU2-BmE that came from silkworm embryonic tissues and BmE cell line was smaller than that between SWAU2-BmE and BmN cell line which originated from ovarian tissue signified the genetic distance between cell lines of same tissues from same species was smaller than that between cell lines of different tissues from same species.Results of isoenzymic analyses showed that expressed varieties, amount and quantity of 4 isoenzymes from SWAU2-BmE cell line, namely, a-esterifiable enzyme, 6-esterifiable enzyme, lactate dehydrogenase, malic dehydrogenase were quite similar to isoenzymic expression charts of its original embryo, however, some diversities still existed. Obvious differences could be seen between the isoenzymic expression charts of SWAU2-BmE and those of other 4 cell lines with the name BmN, Sf9, Hi5, Sf21, which suggested that these isoenzymes could distinguish these different cell lines commendably. In addition, there were some insignificant isoenzymic expression difference between the 15th generation and the 25th generation of SWAU2-BmE cell line.To sum up, over two years' endeavors ,a new cell line of Bombyx mori, namely, SWAU2-BmE has been established through screening out of from embryo tissues with different differentiated level. These cells are small, highly reproducible and duplized. Moreover, they are highly susceptive to BmNPV virus. Because of these distinguished features, this cell line is a relatively desired cell...