JP3035616B1 - Silkworm culture cell line - Google Patents

Abstract

[Summary] [Continuous passage] Bombyx mori cultured cell line capable of growing stably in the temperature range of 25 to 37 ° C There is provided a system which is useful for elucidating the physiological biological functions of insects and which has a simpler constitution of tissue organs of an insect body.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a silkworm cultured cell line which can be stably maintained at an unprecedented high temperature, and more specifically, a silkworm cultured cell line which grows stably in a temperature range of 25 to 37.degree. The present invention relates to a cultured cell line of silkworms derived from silkworms, zozo, yellow waves and black moth.

[0002]

2. Description of the Related Art In elucidating the physiological biological functions of insects,
The establishment of a functional cultured cell line is expected as a system that further simplifies the organization and organization of the insect body. Conventionally, cultivation of silkworm-derived cells has been performed at a temperature in the range of 25 to 27 ° C. in consideration of a temperature suitable for the length of growth of silkworm larvae. However, when long-term culture was continued under these conditions, only cell lines having characteristics capable of proliferating only in this temperature range were obtained, and these cell lines rapidly died when cultured above 30 ° C. Even if selection was repeated assuming that cells cultivable at 37 ° C were included in cells passaged at 25 to 27 ° C, selection of cells cultivable at such a high temperature was impossible.

[0003]

Since the silkworm nucleopolyhedrovirus has a characteristic that it cannot grow at 37 ° C., using a silkworm culture cell line that can grow at a temperature range of 25 to 37 ° C. It can be very useful in controlling virus infection by a simple method of changing the culture temperature and studying the mechanism of infection and proliferation.

[0004] Cultured cells such as mammals other than insects must always be cultured at 37 ° C. Therefore, using a silkworm culture cell line that can be cultured at this temperature, cell fusion with other cultured cells is possible, and it is expected to be promising for various research and industrial progress. Therefore, 25
The usefulness of a cultured silkworm cell line that can grow in a temperature range of up to 37 ° C. is immeasurable, and its establishment has conventionally been desired, but has not been reported so far.

[0005]

Therefore, the present inventors have proposed that
Before immobilizing the characteristics of the cells selected at a temperature in the range of 5 to 27 ° C., it was thought that a mutant strain suitable for culture at 37 ° C. could be obtained. After establishing, the culture temperature was changed to 37 ° C in the subculture, and the surviving cells were further cultured to surprisingly expand cell lines growing in vitro at an unprecedentedly high temperature of 37 ° C. Can be established and completed the present invention.

That is, the present invention relates to a continuously passaged silkworm cultured cell line that stably grows in a temperature range of 25 to 37 ° C. The silkworm culture cell line of the present invention is derived from silkworms, and can be an embryo, an epithelial cell, a muscle cell, a nerve cell, or a fiber if it is a continuous passage that stably grows in a temperature range of 25 to 37 ° C. It may be any of those derived from blast cells, and is not particularly limited, but those derived from embryonic tissue are preferred. In addition, as long as it is derived from silkworm, its variety is not particularly limited, and examples thereof include purple silkworm, Taizo, yellow wave and black moth, but are not particularly limited.

The silkworm culture cell line of the present invention can be suitably used even if it is a polyclonal cell line. However, a cell line obtained by cloning the polyclonal cell by known means and unifying its properties and the like is used. It is also possible. Furthermore, the silkworm cultured cell line according to the present invention can be obtained by the method according to the present invention described below, which was deposited on May 12, 1999 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology.
NISES-BoMo-MK (FERM P-17391), NISES-BoMo-DZ (FERM
P-17389), NISES-BoMo-OH (FERM P-17392), or N
It is also possible to use ISES-BoMo-KG (FERM P-17390). The present invention also provides a method for culturing cells obtained from silkworm embryo tissue in the range of 25 to 27 ° C., establishing primary culture, changing the culture temperature to 37 ° C. in secondary culture, and 25-37 ° C. characterized by culturing
The present invention provides a method for preparing a continuously passaged silkworm cultured cell line that stably grows in the temperature range described above.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The following procedure establishes a continuous passaged silkworm culture cell line according to the present invention which stably grows in a temperature range of 25 to 37 ° C. The individual experimental conditions, for example, silkworm varieties, culture media, subculture intervals, and the like are merely exemplary, and those skilled in the art may change them to the extent that they do not interfere with obtaining the cultured cell line according to the present invention. Can be added.

[0009] Four cultivars of the primary cultured silkworm, purple silkworm, Taizo, yellow wave and black moth, are blue-blued at 25 ° C.
Surface sterilization is performed by soaking in 70% ethyl alcohol for minutes. The sterilized eggs are washed with sterile water and sterile Carlson solution (Carlson, Biol. Bull. 90: 109-121, 194
Transfer to 6), and after incision, remove the embryo from the shell. The embryos are then washed again with a sterile Carlson solution to remove the yolk protein particles. Embryos are separated from the surrounding yolk in Carlson's solution, washed with fresh solution, divided into several pieces,
10% fetal bovine serum (FBS; GIBCO, Grand Island, US
A.) and MGM-448 medium (MiMi
tsuhashi, Zool. Sci. 1: 415-419, 1984). 50
The cells and tissue fragments taken from the individual eggs were transferred to a medium 4 in a tissue culture flask (Sumitomo Bakelite Co., Ltd., 25 cm ² ).
Inoculate in ml and incubate at 25 ° C.

When the subcultured cells begin to grow rapidly in the primary culture, they are split into two flasks for the secondary culture. One vessel is maintained at 25 ° C as usual and the other is maintained at 37 ° C. 10
Exchange 2 ml of medium with fresh medium at intervals of once every two weeks for a period of about a month. Thereafter, the transplanted cells at the bottom of the container begin to divide rapidly until they grow to saturation on the flask surface. The cells are detached from the flask by flushing the medium with a pipette over the cells and inoculating a new flask. The first to tenth subcultures are performed every 10 to 14 days, and after the eleventh subculture, half of all cells in one flask are subcultured to a new flask once a week. The cultured cell system according to the present invention obtained as described above characterizes cells, for example, as follows, but the procedure for characterizing does not limit the present invention in any way.

[0011] 1. When the cell morphology has been passaged approximately 250 times, the morphology of the cells is examined, and photographs of the cultured cells are taken with a phase-contrast microscope. 2. The cell population is counted every two days after subculture to measure the growth of proliferating cells.

[0012] 3. At the next planting of karyotype 40 times, the method of Early (TCA Manual 1: 31-3
5, 1975), and analyze the karyotype. Since the nucleus contains many microchromosomes,
It is difficult to accurately count chromosomes, and the results are usually variable (Mitsuhashi, Appl. Entomol. Zoo)
l. 1: 103-104, 1966; Biomedical Research. 2: 599-60
6, 1981; Develop., Growth and Differ. 23: 63-72, 19
81; Appl. Entomol. Zool. 18: 533-539, 1983; Zool. Sc
i. 1: 415-419, 1984; Appl. Entomol. Zool. 30: 75-82,
1995; Jpn. J. Ent. 64: 692-699, 1996; Mitsuhashi and Shozawa, Develop., Growth and Differ. 27: 599-6
06, 1985; Mitsuhashi and Inoue, Appl. Entomol. Zo
ol. 23: 488-490, 1988).

[0013] 4. Enzyme activity isozyme detection kit (Authentikit, Corning Sci
ence Products, Corning Glass Works, MA, USA) using phosphoglucose isomerase (PGI: ECno.
5.3.1.9), phosphoglucose mutase (PGM: EEno.2.
7.5.1), isocitrate dehydrogenase (ICD: ECno.
1.1.1.42) and the activity of glucose-6-phosphate dehydrogenase (G6PD: ECno.1.1.1.49). In this analysis, 1 × 10 ⁷ cells cultured at 25 ° C. or 37 ° C. for 2 days are used.

[0014] 5. Virus-resistant silkworm nuclear polyhedrosis virus, BmP6E strain (Kobayashi et al., I
n: HH Hagedon, JG Hildebrand, MG Kidwell and JH Law (eds.), Molecular Insect Science. Plenum,
(New York, NY. 325pp, 1990) using a 96-well multi-well plate to try to infect the cell line according to the invention and its clonal cell line. After seeding 3 × 10 ⁴ cells in each well, NISES-BoMo-15AIIc inoculated with BmP6E (Inoue et al., Bull. Natl. Inst.
Culture supernatant 25 of Seric. Entomol. Sci. 1: 13-25, 1990)
Add μl. Two hours after inoculation, the virus suspension is taken, replaced with fresh medium containing 10% FBS, and the cells are incubated at 27 ° C. Seven days after BmNPV infection, cells in each well are examined for the presence of polyhedra.

[0015] 6. 8 × 10 ⁵ cells are seeded in each flask into effect medium 4ml of medium exchange frequency. To examine the effect of 37 ° C. on both cell growth and media condition, the following two experiments are performed. That is, in the first experiment, half of the medium is replaced with a fresh medium every day, and in the second experiment, the entire medium is replaced with a new medium every four days.

[0016]

The present invention will be described in detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 Establishment of Cultured Cell Line from Purple Silkworm Embryo Tissue The embryonic tissue derived from primary cultured purple silkworm was cultured to establish a cell line. The culture started on June 22, 1991. Various types of cells gradually migrated from the explant tissue. During the first three months, fibroblasts radiating from the explant formed a network. This network then formed large and small round cells, strong adherent cells and muscle-like cells showing contraction. The most common cell type was epithelial-like cells attached to flasks. In addition, a network formed by fibroblast-like cells was also observed. This outgrowth of cells is common in the culture of insect embryo tissue (Dwig
ht, In Vitro Cell Dev. Biol. 31: 91-93, 1995; Mitsuh
ashi and Maramorosch, Contrib. Boyce Thompson Ins
t. 22, 435-460, 1964; Mitsuhashi, Appl. Entomol. Z
ool. 8: 64-72, 1973; Mitsuhashi, Biomedical Researc
h. 2: 599-606, 1981; Develop., Growth and Differ. 2
3: 63-72, 1981; Imanishi and Ohtsuki, J. Serial. Sc
i. Jpn. 57: 184-188, 1988; Inoue and Mitsuhashi,
J. Seric. Sci. Jpn. 53: 108-113, 1984; Ninagi et al., 3r.
d International Cell Culture Congress (Sendai, Japa
n), Abstruct 69, 1985; Takahashi et al., Develop., Grow
th andDiffer. 22: 11-19, 1980). Approximately 12 months after the start of the primary culture, the vessels were completely covered with cells and some were floating in the medium.

Subculture During the primary culture , on September 16, 1992, the cells began to increase rapidly. Therefore, the secondary culture was started on September 22, 1992.
After the start of the primary culture, it took 457 days to start the secondary culture of purple silkworm cells. Cells were detached by pipetting and transferred to several flasks. During the early stages of subculture, cells grew very slowly. At this stage, some of the cells were transferred from 25 ° C to 37 ° C. The high temperature killed the majority of cells, but the number of surviving cells gradually increased, and cells adapted to 37 ° C. were able to grow normally. After the cells had grown to saturation on the flask surface, the cells no longer increased and began to die. When cultured at 37 ° C., cells must be subcultured every 4 days if the medium is not changed. After the fifteenth generation, the growth rate appeared to have stabilized, and one fifth of the total cells in one flask were subcultured to another flask once a week. The cell line that was passed over 100 times was named NISES-BoMo-MK.
This cell line was adherent, but was easily detached by pipetting. Based on its shape, the cell line appeared to be epithelial cells. Silkworm varieties are weak to 37 ° C and die easily (Yamamoto et al., J. Seri
c. Sci. Jpn. 60:52, 1990), but this cell line from the silkworm embryo survived at 37 ° C. This means that the optimal temperature for larval survival is different from the optimal temperature for cultured cell survival.

Example 2 Preparation of Clone Strain Using the NISES-BoMo-MK obtained in Example 1, two types of clone strains, NISES-BoMo-MKc2 and NISES-BoMo-MKc4, were prepared. Made by Seaplak agarose (SeaPlaque)
Was overlaid on a plastic petri dish, a small number of cells were spread thereon, and cultured, and a cell colony of a clone was recovered.

Example 3 Establishment of Cultured Cell Line from Large Embryonic Tissue In the same manner as in Example 1, a cultured cell line was established from large embryonic tissue. The primary culture culture started on July 6, 1991. Various types of cells gradually migrated from the explant tissue. During the first three months, fibroblasts radiating from the explant formed a network. Then, this network is large and small circular cells,
Strongly adherent cells and muscle-like cells showing contraction were formed. The most common cell type was epithelial-like cells attached to flasks. In addition, a network formed by fibroblast-like cells was also observed. Approximately 12 months after the start of the primary culture, the vessels were completely covered with cells and some were floating in the medium.

Subculture During the primary culture , on September 16, 1992, the cells began to increase rapidly. Therefore, the secondary culture was started on September 22, 1992.
After the start of the temporary culture, it took 471 days before the start of the secondary culture of the large-scale cells. Remove cells by pipetting,
Transferred to several flasks. During the early stages of subculture, cells grew very slowly. At this stage, some of the cells were transferred from 25 ° C to 37 ° C. The high temperature killed the majority of cells, but the number of surviving cells gradually increased, and cells adapted to 37 ° C. were able to grow normally. After the cells had grown to saturation on the flask surface, the cells no longer increased and began to die. When cultured at 37 ° C., cells must be subcultured every 4 days if the medium is not changed. After the fifteenth generation, the growth rate appeared to have stabilized, and one fifth of the total cells in one flask were subcultured to another flask once a week. The cell line that was passed over 100 times was named NISES-BoMo-DZ.
This cell line was adherent, but was easily detached by pipetting. Based on its shape, the cell line appeared to be epithelial cells.

Example 4 Establishment of Cultured Cell Line from Yellow Wave Embryo Tissue In the same manner as in Example 1, a cultured cell line was established from yellow wave embryo tissue. Primary culture Yellow wave culture started on July 6, 1991. Various types of cells gradually migrated from the explant tissue. During the first three months, fibroblasts radiating from the explant formed a network. Then, this network is large and small circular cells,
Strongly adherent cells and muscle-like cells showing contraction were formed. The most common cell type was epithelial-like cells attached to flasks. In addition, a network formed by fibroblast-like cells was also observed. Approximately 12 months after the start of the primary culture, the vessels were completely covered with cells and some were floating in the medium.

Subculture During the primary culture , on September 16, 1992, the cells began to increase rapidly. Therefore, the secondary culture was started on September 22, 1992.
After the start of the temporary culture, it took 471 days to start the secondary culture of the yellow wave cells. Remove cells by pipetting,
Transferred to several flasks. During the early stages of subculture, cells grew very slowly. At this stage, some of the cells were transferred from 25 ° C to 37 ° C. The high temperature killed the majority of cells, but the number of surviving cells gradually increased, and cells adapted to 37 ° C. were able to grow normally. After the cells had grown to saturation on the flask surface, the cells no longer increased and began to die. When cultured at 37 ° C., cells must be subcultured every 4 days if the medium is not changed. After the fifteenth generation, the growth rate appeared to have stabilized, and one fifth of the total cells in one flask were subcultured to another flask once a week. The cell line that was passed over 100 times was named NISES-BoMo-OH.
This cell line was adherent, but was easily detached by pipetting. Based on its shape, the cell line appeared to be epithelial cells.

Example 5 Establishment of Cultured Cell Line from Black Moth Embryo Tissue In the same manner as in Example 1, a cultured cell line was established from yellow wave embryo tissue. Culture of the primary culture black moth started on June 22, 1991. Various types of cells gradually migrated from the explant tissue. During the first three months, fibroblasts radiating from the explant formed a network. Then, this network is large and small circular cells,
Strongly adherent cells and muscle-like cells showing contraction were formed. The most common cell type was epithelial-like cells attached to flasks. In addition, a network formed by fibroblast-like cells was also observed. Approximately 12 months after the start of the primary culture, the vessels were completely covered with cells and some were floating in the medium.

Subculture During the primary culture , on September 16, 1992, the cells began to increase rapidly. Therefore, the secondary culture was started on September 22, 1992.
After the start of the temporary culture, it took 457 days to start the secondary culture of the black moth cells. Remove cells by pipetting,
Transferred to several flasks. During the early stages of subculture, cells grew very slowly. At this stage, some of the cells were transferred from 25 ° C to 37 ° C. The high temperature killed the majority of cells, but the number of surviving cells gradually increased, and cells adapted to 37 ° C. were able to grow normally. After the cells had grown to saturation on the flask surface, the cells no longer increased and began to die. When cultured at 37 ° C., cells must be subcultured every 4 days if the medium is not changed. After the fifteenth generation, the growth rate appeared to have stabilized, and one fifth of the total cells in one flask were subcultured to another flask once a week. The cell line that was passed over 100 times was named NISES-BoMo-KG.
This cell line was adherent, but was easily detached by pipetting. Based on its shape, the cell line appeared to be epithelial cells.

Comparative Example 1 NISE derived from silkworm day 125 embryo cultured at 25 ° C. for a long time
S-BoMo-J125K2 cell line (Imanishi and Tomita, JARQ. 2
6: 196-202, 1992) were used as comparative examples. Characterization 1 of the cell. Cell morphology After passage about 250 times in the secondary culture, NISES-BoMo-MK, NISES-BoMo-DZ, N
The morphology of each cultured cell line of ISES-BoMo-OH and NISES-BoMo-KG was examined. The results are shown in FIG. As shown in FIG.
The cultured cell lines according to the present invention were all adherent cells of an epithelial cell line having a uniform size and shape.

[0026] 2. Proliferated four cell lines, NISES-BoMo-MK, NISES-BoMo-D
Z, 25 of NISES-BoMo-OH and NISES-BoMo-KG cells
Growth at 28 ° C, 28 ° C and 37 ° C was examined. As an example, FIG. 2 shows the growth curves of NISES-BoMo-OH according to the present invention and NISES-BoMo-J125K2 of Comparative Example 1. The initial density was 2.0 × 10 ⁵ cells / ml (medium), and cell growth was measured every two days after subculture. When cells were cultured at 25 ° C., the interval between subcultures was 7 days.

As is apparent from FIG.
The species cell lines showed almost the same growth rate at 25 ° C., 28 ° C. and 37 ° C., but the NISES-BoMo
The -J125K2 cell line showed no growth at 37 ° C and all died. Suitor (Exptl. Cell Res. 44: 572-578, 196
6) reports that the cell density of clones derived from the Grace's mosquito cell line is lower than the initial density when cultured at 34 ° C., but the cell line established by the present inventors is 37.
° C could be adapted. Possibly, cells with a very high optimal temperature for their growth were selected.

[0028] 3. Were cultured in karyotype 25 ° C. and 37 ℃ NISES-BoMo-MK, N
The karyotype of ISES-BoMo-DZ, NISES-BoMo-OH, and NISES-BoMo-KG cells was examined up to approximately the 40th generation. The results are shown in FIG. The data in the figure shows the average of 50 chromosome samples.
The number of diploids is 28 chromosomes. As shown in FIG.
Although the karyotype of these cell lines was typical of lepidopteran cell lines, the distribution pattern of chromosome numbers varied with the temperature at which the cells were cultured. Cells cultured at 25 ° C. had fewer chromosomes than those cultured at 37 ° C. The number of chromosomes in the cell line ranges from 90 to 1 for cells cultured at 25 ° C.
It ranged from 40 to 100 to 265 for cells cultured at 37 ° C.

Mitsuhashi (Jap. J. appl. Entomol. Zoo
l. 9: 107-114, 1965) suggest that when embryo cultures of Nephotettix cincticeps are cultured in vitro, giant cells are often seen, which is expected to be polyploid. In the cell line according to the present invention, more giant cells appeared more frequently in cells cultured at 37 ° C than those cultured at 25 ° C.

[0030] 4. Enzyme activity The enzyme activity was measured, and the cultured cell lines according to the present invention of Examples 1 to 4 and the NISES-
BoMo-J125K2 cell line (Imanishi and Tomita, JARQ. 26:
196-202, 1992). The conditions for measuring the enzyme activity are as follows. The enzyme extract was added to the cultured cells, and the cells were ground using a homogenizer. The obtained cell extract is electrophoresed after adding several microliters to each lane of the agarose film gel, and the separated band is stained with an enzyme substrate to confirm the position and number of the band. These methods were performed as described in the enzyme detection kit manual. The voltage of the electrophoresis apparatus is a constant voltage of 100 volts, and the electrophoresis time is 25 minutes.

Isozyme detection kit (Authentikit
Using these lines, these five cell lines were identified as silkworm cell lines. NISES-BoMo-MK, NISES-B according to the present invention
oMo-KG, NISES-BoMo-OH, and NISES-BoMo- of Comparative Example 1
Phosphoglucomutase (PG) in each of J125K2
M), phosphoglucose isomerase (PGI), isocitrate dehydrogenase (ICD), and glucose-6
-The influence of the culture temperature (25 ° C and 37 ° C) on the enzyme activity of phosphate dehydrogenase (G6PD) was examined. Table 1 shows the results.

[0032]

[Table 1]

In the table, "-" indicates that all cells were killed, and the NISES-BoMo-J125K2 cell line of Comparative Example 1 could not survive at 37 ° C. An enzyme extract was prepared from about 1 × 10 ⁷ cells two days after subculture. As can be seen from the results in Table 1, the activity of enzymes other than PGM, PGI and G6PD, ie ICD, was greater than 25 ° C in all cell lines at 37 ° C.
C was high. This indicates that ICD plays an important role in the citrate cycle, and that ICD activity is weakened under abnormal conditions such as 37 ° C. Furthermore, even though the cell lines were derived from silkworm embryo tissue, these cell lines showed different activities with the four enzymes.

[0034] 5. NISES-B inoculated with virus-resistant silkworm nuclear polyhedrosis virus (BmNPV)
oMo-MK cell line and its clone cell line NISES-
The effect of the culture supernatant of the cells (NISES-BoMo-15AIIc) on the polyhedron-forming ability in BoMo-MKc2 and NISES-BoMo-MKc4 was examined. Table 2 shows the results.

[0035]

[Table 2]

The numerical values in the table indicate the number of wells where polyhedra were observed in the cells / the number of wells examined. As shown in Table 2, the NISES-BoMo-MK cell line according to the present invention, and its clone cell lines NISES-BoMo-MKc2 and NISES-BoMo-
In MKc4, the silkworm nucleopolyhedrovirus did not form a polyhedron even in the culture supernatant of infected cells and its diluted solution (10 ^-1 to 10 ^-5 ), indicating virus resistance. On the other hand, in the well-known culture cell lines NISES-BoMo-15AIIc cells and Bm5 cells used as a control, polyhedra were formed in all wells. Other cell lines according to the invention, NISES-BoMo-KG, NISES-BoMo-OH and NISES-BoMo-D
Z also did not form a polyhedron (results not shown). This revealed that the cultured silkworm cell line according to the present invention was resistant to the silkworm nuclear polyhedrosis virus.

[0037] 6. Influence of medium exchange frequency The effect of medium exchange frequency on cell growth when cells were cultured at 37 ° C was examined using NISES-BoMo-OH cells of Example 4. With a four-day subculture interval, after four days, the cells began to die if the medium was not changed. When half of the medium was changed every day, the cells continued to grow until day 8 after subculture, and the number of cells increased from 2 × 10 ⁵ to 1.3 × 10 ⁶ cells / ml of the medium. After the entire population had grown to saturation on the flask surface, without subculture, cells no longer increased and began to die (FIG. 4). MGM-448
It is concluded that any material in the medium can easily degrade at 37 ° C. When cells were cultured at 37 ° C., it was found that it was better to change the medium every day.

As described above, in the present invention, 37
Four cell lines from silkworm embryos adapted to ° C were established.
These cell lines were kept at 25 ° C. and 28 ° C.
At 37 ° C. and at a similar rate. When cultured at 25 ° C., the number of chromosomes in the cells is 3
It was smaller than at 7 ° C. The activity of enzymes other than PGM, PGI and G6PD, ie ICD, was higher at 37 ° C than at 25 ° C in all cell lines. Furthermore, NISES-BoMo-D
Z, NISES-BoMo-OH, NISES-BoMo-KG and NISES-BoMo-MK
Cell line, and its clone cell line, NISES-BoMo-M
Kc2 and NISES-BoMo-MKc4 did not form the polyhedron of silkworm nuclear polyhedrosis virus.

[0039]

As described above in detail, the present invention makes it possible to obtain a continuously passaged silkworm cultured cell line that can stably grow in a temperature range of 25 to 37 ° C., which has never existed before. The present invention has provided a system which is useful for elucidating the physiological biological functions of worms and which has a simpler structure of the organs of the insect body. By using the silkworm cultured cell line according to the present invention, it is possible to control the infection of silkworm nuclear polyhedrosis virus that cannot grow at 37 ° C. and study the mechanism of infection / growth by a simple method of changing the culture temperature. Therefore, it is expected that the regulation of the expression of the gene introduced into the cell will be elucidated.

In addition, using the silkworm cultured cell line according to the present invention, compared with other cultured cells such as mammals which need to be constantly cultured at 37 ° C., the physiological organisms of insects and other cell lines were compared. It can be used to compare functions. In addition, cell fusion with other cultured cells is also possible, which can contribute to various researches and industrial progress such as regulation of expression of foreign genes. Furthermore, the cloning of the cultured cell line according to the present invention unifies the properties and provides a cultured cell line which is more useful as a material for various studies, such as elucidation of biological functions of insects.

[Brief description of the drawings]

[Figure 1] NISES-BoMo-MK, NISES-BoMo-KG, NISES-
It is a phase contrast micrograph of BoMo-DZ and NISES-BoMo-OH.

FIG. 2 shows the effect of temperature on cell growth.

FIG. 3 shows a distribution pattern of the number of chromosomes per cell.

FIG. 4 shows the effect of medium exchange on cell growth at 37 ° C. ■: Change half of medium every day ●: Change half of medium every 4 days

Claims (6)

(57) [Claims] 1. A continuously passaged silkworm cell line that grows stably in the temperature range of 25 to 37 ° C. 2. The cultured silkworm cell line according to claim 1, which is derived from a silkworm embryo tissue. 3. The cultured silkworm cell line according to claim 1, wherein the cultured silkworm cell line is derived from purple silkworm, large artificial, yellow wave or black moth. 4. The cultured silkworm cell line according to claim 1, which is a cloned cell line. 5. NISES-BoMo-MK (FERM P-17391), NISES
-BoMo-DZ (FERM P-17389), NISES-BoMo-OH (FERM P-17
The cultured silkworm cell line according to claim 1, which is selected from the group consisting of 392) and NISES-BoMo-KG (FERM P-17390). 6. The method according to claim 6, wherein the cells obtained from the silkworm embryo tissue are 25
2. The cultured silkworm cell according to claim 1, wherein the cultured cell is cultured at a temperature of up to 27 ° C., a primary culture is established, the culture temperature is changed to 37 ° C. in a secondary culture, and the surviving cells are further cultured. How to create a system.