In VitroCell.Dev.Biol.28A:779-781.November-December1992 © 1992TissueCultureAssociation 0883-8364/92 $01.50+0.00

BASIC CONDITIONS FOR THE DRUG SELECTION AND T R A N S I E N T EXPRESSION

IN THE

CULTURED

CELL

LINE

OF BOMBYX

GENE

MORI

KAZUHIRO OKANO, NAOKO MIYAJIMA, NAOKO TAKADA, MASAHIKO KOBAYASHI, Arid HIDEAKI MAEKAWA

Department of Agrobiology, Faculty of Agriculture, The Universityof Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo I 13, Japan (K. 0., M. K.) and Department of Technology, National Institute of Health, 2-10-35, Karniosaki, Shinagawa-ku, Tokyo 141, Japan (N. M., N. T., H. M.) (Accepted 16 June 1992)

SUMMARY We established basic conditions for transient gene expression and selection of antibiotics in the cultured cell line of silkworm, Bombyx mori, by use of the promoter of the heat shock protein (hsp70) gene of Drosophila melanogaster. The control promoter (hsp70) promoted the expression of chloramphenicol acetyltransferase (CAT) gene ligated at the downstream, dependent on the orientation of the promoter in the silkworm cell. The cell line is able to be supplied for the promoter assay of the silkworm genes. The concentration for the drug selection was determined as 0.75 mg/ml on neomycin analog, G418 (geneticin).

Key words: G418; drug selection; transfection; transient gene expression; SES-Bm-I 30T; silkworm; Bombyx mori. selected in our laboratory as SES-Bm-1 30T with more adhesive properties than the original 1 30 line. The culture medium uses IPL-41 supplemented with 5% fetal calf serum (Filtron Pty., Altona, Victoria, Austraha), prepared according to R. H. Goodwin (personal communication, Institute of Kyodo Shiryo Co., Ltd.). However, in some cases, fetal calf serum is not suitable for B. mori cell lines and then several lots of serum should be checked for compatibility before use. For the transfection, cells of about 1 × 105 were freshly prepared from the cultured fraction about l 0 6 cells before use. The Drosophila hydei cell line KUN-DH33 (a gift from Dr. Miyake, Mitsubishi Kasei Institute of Life Sciences) (5), was used as a control. Chemicals. Neomycin analog, G418 (geneticin), was purchased from GIBCO BRL (Gaithersburg, MD). lgC-Chloramphenicol of Amersham International plc(Little Chalfom, Buckinghamshire, England) was used. Restriction enzymes were purchased from Boehringer Mannheim GmbH-Biochemica (Mannheim, Germany) and Takara Shuzo (Kyoto, Japan). Other reagents were from Wako Chemicals (Ohsaka, Japan). Plasmid DNA. pFb(-860/+67)CAT (8) contains a CAT gene and SV40 poly(A) addition region, and in the promoter of the reporter gene the region derived from the heat shock protein 70 gene (hsp70) of D. melanogaster was substituted for that of the B. mori fibroin gene. In order to check whether the orientation of promoter is recognized or not, the both directions of the hsp70 promoter are constructed. For transfection, 5 #g of plasmid DNA was added per flask containing 3 ml IPL-41 medium by use of a TransPhect kit (Pharmacia LKB Biotechnology, Uppsala, Sweden). The medium was changed after growth overnight and cultured for all additional 48 h. After harvesting, the cells were washed two times with B. mori saline (1.12 g NaCI, 24 mg KCI, and 20 mg NaHCO3 are dissolved with 100 ml H20 followed by addition of 27 mg CaC12) and resuspended in 100 #1 of 0.25 M Tris (pH 7.8). Freezing and thawing were repeated by three cycles and the lysate was added to a reaction mixture containing l*C-chloramphenicol followed by extraction with ethyl acetate. CAT activity was detected as a acetylated chloramphenicol by thinlayer chromatography (TLC) using chloroform:methanol (95:5) as solvent (2). When quick data was needed, an AMBIS radioacdvity detector was used while normal detection by autoradiograph.

INTRODUCTION The silkworm, Bombyx mori, has been used for studying many aspects of genetics, physiology and sericuhure in Japan for more than a hundred years. B. mori has unique and convenient characteristics for research in the ability to control the early development of egg and larval stages. However, some systems enabling the use of genetic engineering techniques have been especially delayed compared to other widely used experimental animals in which a transient or stable gene expression system has been developed (1). In this situation, cultured cells must be adapted or improved to use for the introduction of gene whose expression is easily detected in an interest and convenient system. The neomycin analog, G418 (geneticin), has been widely used as the selection reagent in several species (4). In insects as Drosophila, the resistant concentration ( 1 - 2 mg/ml) is usually higher than that of mammals (0.1-0.8 mg/ml) (3,7). Therefore, we first determined the concentration for the selection of transformants by the measurement of the cell toxicity in several different concentrations of G418. An expression system has already been established for B. mori in which a gene injected into a fertilized egg is transiently expressed until hatching (8). However, we have not yet established a transient expression system in cultured ceils. As one basic approach, we tested the promoter activity of a Drosophila melanogaster heat shock protein gene (hsp70) in cultured Bombyx cells transfected with a plasmid using the chloramphenicol acetyltransferase gene (CAT) as a reporter. In the present paper, we show the first observation of transient gene expression using G418 toxicity for drug selection as a basic assay system in B. mori cultured cell lines.

RESULTSAND DISCUSSION

MATERIALSAND METHODS

Cells were cultured for 14 days, and at four interval points cell density was determined by counting the cell number while the initial

Cells. The cell lines, SES-Bm-I 30 and SES-Bm-e 21, were established from 10 day embryos (6), and an aliquot of SES-Bm-1 30 was 779

780

OKANO ET AL.

lxlO 6~

Omg/ml

0.25mg/ml ~ lx105. ~ 5x10 4 ~ - • 0

T 3



, • , . , _. 6 9 12 Culture time (days)

0.5mg/ml 0.75mg/ml 1.0, 1.25, 1.5mg/ml

Fie. 1. G418 toxicityin the cultured B. mori cell line, SES-Bm-1 30T. 0.5 × 105 cells were prepared and cultured in 3 ml of IPL-41 medium supplemented with 5% fetal calf serum and 0-1.5 mg/ml G418. The cell number was determined for 14 days using light microscopy. Horizontal, culture time (days): vertical, cell number (cells/ml). 0 mg/ml, O; 0.25 mg/ml, O; 0.5 mg/ml, A; 0.75 mg/ml, A. Cell number at 1.0, 1.25 and 1.5 mg/ml; I , are the same as the bottom line.

cell number is 0.5 × I 0 s cefls/ml. The doubling time of this cell line was determined as about 60 h as shown in Fig. 1 (0 mg/ml G418). The same doubling time was obtained in a different way by measuring thymidine incorporation (data not shown). Then the de-

crease in cell number dependant on the increase of G418 concentration in a range of 0 to 1.5 mg/ml was observed. As shown in Fig. 1, at concentrations of 1.0, 1.25 and 1.5 mg/ml, we could observe broken ceils in the culture even on the first day after addition of the drug, until finally almost all cells were dead. Cell growth was inhibited up to 63% at 0.25 mg/ml of G418, while at 0.5 mg/ml and 0.75 mg/ml, it inhibited to 85% and 90% of the control, respectively. Therefore, 0.75 mg/ml is chosen as a suitable concentration for drug selection. However, we recommend using a lower concentration for G418 selection for the more efficient recovery of transformed cells. In order to construct a transformation system, it is important for us to test whether silkworm cell lines are to be used for a transient gene expression system. The original cell line, SES-Bm-1 30, was maintained in an aggregated form (personal communication, O. Ninaki), but revealed the property of weak adhesion to the flask, which is not good for transfection. The repeated selected ceil line has the more adhesive ability than the original and it seemed that DNA-calcium phosphate complexes were incorporated well into the cell. In order to construct a control plasmid for checking the expression system, we chose the heat shock promoter, hspT0, ofD. melanogaster, because of its high promoter activity in several species. In order to assay the dependence of expression on promoter orientation, both directions of the promoter were inserted into a plasmid containing the chloramphenicol acetyltransferase (CAT) reporter

BSHB S

i

Normal

S

CAT IA, I

BSHX Re v e r s e

B

SBH

liii =:=:: iiii

iliiii

B

S

CAT

A

FIg. 2. Plasmids for transient expression assay constructed with both orientations of the Drosophila hsp70 promoter. The plasmid was constructed with the hsp promoter from puchsTrA2-3 (2), polylinker, and part of CAT from pKK232-8 (purchased from Pharmacia), and part of CAT and SV40 terminator from pFb(-860/+67)CAT designated (,4) on Figure (8).

TRANSIENT EXPRESSION AND DRUG SELECTION IN BOMBYX MOR1 CULTURED CELLS

1 234

5 6

78

3-AC 1 -AC

CM

781

enzyme, CAT. Accordingly, this hsp promoter is useful as an appropriate promoter to assay system transient expression and as a control promoter to test other unknown promoters because of its dependency on species and tissue specificity (data not shown). In a different cell line, SES-Bm-e 21, which has more adhesive properties, similar to fibroblasts, the same level of CAT activity was also detected by use of the hsp promoter. As a final note, we point out that in B. mori cell lines, conditions for transfecfion are evidently somewhat different from the near confluent condition of mammal cell systems because lower cell concentrations (about 105 cells/ml) give rise to higher activity of CAT (data not shown).

ACKNOWLEDGEMENTS We appreciate Drs. M. R. Goldsmith and O. Ninaki critical reading and encouragement and Mr. Y. Kikuchi and Miss Kanai for the supplement of the medium and cells.

REFERENCES

FIG. 3. Expression of the hsp promoter with CAT reporter gene in cultured cell line. The plasmid constructed with the hsp promoter, CAT gene and SV40 terminator was added into each cultured cell line by transfection. After overnight storage, the medium was changed and cells were cultured for 48 h more. The harvested cells were assayed for the detection of CAT activity. Lane 1, control activity by chloramphenicol acetyltransferase; lane 2, not added plasmid; lanes 3, 5 and 7, the normal direction of hsp promoter; lanes 4, 6 and 8, the opposite direction ofhsp promoter. Lanes 3 and 4 are in KUN-DH33 cells; lanes 5 and 6 are for SESoBm-e 21 cells; and lanes 7 and 8 are for SES-Bm-1 30T cells. CM, not acetylated 14C-ehlorampbenicol; 1-AC, position-1 acetylated; 3-AC, position-3 acetylated chloramphenicol. gene and the SV40 termination region (Fig. 2). Expression of CAT activity occurred only in the normal direction as shown in Fig. 3. These experiments show that the transfected plasmid DNA was incorporated, expressed as mRNA, and finally translated to the

1. First, N. L.; Haseltine, F. P., ed. Transgenic animals. ButterworthHeinemann, Stoneham; 1991. 2. Gorman, C. M. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2:10441051; 1982. 3. Maisonhaute, C.; Echalier, G. Stable transformation of Drosophila Kc cells to antibiotic resistance with the bacterial neomycin resistance gene. FEBS Lett. 197:45-49; 1986. 4. Michel, K. Gene transfer and expression, a laboratory manual. New York: Stockton Press; 1990:103-113. 5. Miyake, T.; Mae, N., et al. Protein of virus-like particles by the transposable genetic element, copia of Drosophila melenogaster. Mol. Gen. Gent. 207:29-37; 1987. 6. Ninaki, O.; Takada, N., et al. Gene analysis by blot hybridization on the silkworm, Bombyx mori, cell lines. Invertebrate cell system applications. Boca Raton: CRC Press; 1:143-149; 1989. 7. Rio, D. C.; Rubin, G. M. Drosophila melanogaster cells with a dominant selectable marker. Mol. Cell. Biol. 5:1833-1838; 1985. 8. Tamura, T.; Kanda, T., et al. Transient expression of CAT genes injected into early embryos of the domesticated silkworm Bombyx mori. Jap. J. Genetics. 65:401-410; 1990.

Basic conditions for the drug selection and transient gene expression in the cultured cell line of Bombyx mori.

We established basic conditions for transient gene expression and selection of antibiotics in the cultured cell line of silkworm, Bombyx mori, by use ...
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