GB2159821A - Transformation of lower eukaryotic cells - Google Patents

Abstract

A process which permits DNA transformation of a lower eukaryotic cell, such as Dictyostelium, by feeding the amoebae with chloramphenicol-induced plasmid in a bacteria carrier, such as E. coli, is described. The plasmid transforms the lower eukaryotic cell at a high frequency and integrates into the nuclear genome. The method is efficient and simple to perform.

Description

SPECIFICATION Transformation of lower eukaryotic cells The present invention relates to a method for the production of macromolecules by genetic engineering. More particularly, the invention relates to the use of Dictyostelium, or other lower eukaryotic cell, as a vehicle for the production of foreign macromolecules by genetic engineering methods.

In accordance with the present invention, a method is provided for the production of macromolcules (proteins) by genetic engineering. The method permits DNA transformation of a simple eukaryotic cell, particularly Dictyostelium, by feeding amoebae with chloramphenicol induced plasmid in a bacteria carrier, such as E. coli. The plasmid transforms the lower eukaryotic, such as Dictyostelium, at a high frequency and integrates into the nuclear genome.

The described method is efficient and simple to perform.

The present invention, therefore, establishes that Dictyostelium and other lower eukaryotic cells can be used as an efficient vehicle for production of foreign macromolecules (proteins) by genetic engineering methods. It is theorized that by using the presently described feeding system any organism that is able to "eat" bacteria can be transformed. Thus, the feeding system for cells capable of eating bacteria represents a significant simplification for transformation of this type of organism. The organism is transformed by its natural feeding system under natural conditions. No new parameters have to be introduced. Also, by the presently described method the plasmid DNA is protected at all times since it is either inside the bacteria or inside the recipient cell.For this reason, time is not a critical parameter and the organisms can be fed with bacteria containing plasmid for a number of days (in other transformation systems, the DNA of the plasmid is in contact with the organism for about 1 hour at 4"C), thereby greatly increasing the number of copies of the plasmid DNA introduced. This is believed to be one of the reasons for the increased yield of transformation.

According to this invention, it has also been discovered that a promotor which functions in mammalian cells, but not-in yeast cells (Kiss, G. B., R. E. Pearlman, K. V. Cornish, J. D.

Friesen, and V. 0. Chan,-1982, J. Bacteriol., 149:542-547), functions in the lower eukaryotic cells such as Dictyostelium. This feature plus other properties of Dictyostelium amoebae and other lower eukaryotic cells makes it comparable or even superior to yeast for commercial in vitro production of human and other eukaryotic gene products by recombinant DNA methods.

An additional important discovery of the present invention is that if the plasmid present in the bacteria fed contains DNA homologous to the DNA of the recipient organism, a more stable and efficient transformation will occur due to integration of the plasmid DNA into the recipient genomic DNA.

Dictyostelium or other lower eukaryotic cells are, therefore, an important alternative to prokaryotic systems for the processing and production of macromolecules of eukaryotic origin by genetic engineering methods. These systems will avoid the problems observed in prokaryotic systems due to their lack of splicing of RNA and post-transcriptional modifications of the protein produced (i.e. glycosilation, phosphorylation, etc.). The system can be used for the production of vaccines, hormones, Interferon, regulatory proteins, enzymes, and others.

DESCRIPTION OF PRESENTLY PREFERRED EMBODIMENT Brief Description of Drawings Figure 1 illustrates the recombinant plasmid pBM 7.6 which carries Dictyostelium DNA. This plasmid is very efficient in transforming Dictyostelium and provides stable transformants after insertion into the genome through homologues recombination. In detail, Fig. 1 shows the structure of plasmid pBM 7.6 used for feeding transformation of Dictyostelium discoideum. The thick lines represent regions of pAG60 that are homologous to pBR322. The dashed line terminated by Pvu II sites corresponds to the region of PAG60 that has the herpes simplex virus TK promoter and the bacterial kan gene. The narrow line demarcated by Hind Ill and Bam HI denotes the Dictyostelium DNA insert.

Figure 2 illustrates the mammalian cell transformation vector, pSV2-neo which does not carry Dicktyostelium sequences. This plasmid transforms at a lower efficiency.

Figure 3 is a graph which illustrates the effect of G-41 8 on the growth of 6-days feeding experiment with chloramphenicol amplified pBM 7.6. In detail, Fig. 3 shows the effect of G-418 on the growth of amoebae transformed by feeding. Amoebae, fed for 6 days with LA102 contained chloramphenicol amplified pBM 7.6, were divided into two aliquots, one portion cultured in the absence of G-418, (A), and the other in the presence of 40 'ig G-418 per ml, (B). As a control, wild type cells, fed with bacteria free of plasmid, were treated in the same way, (C) and (D).

Working Example Of A Protocol For Dictyostelium Discoideum Transformation A. Materials Utilized in Protocol: (1) E. coli B/r grown to saturation in NZY broth, NZ broth, or L-broth.

NZY broth: 10 gm N-Z-amine A (Sheffield Products, POB 398, Memphis, TN 38101); 5 gm NaCI; 2 gm MgCI2.7H2O; 5 gm yeast extract (Difco); and H2O to 1 liter.

NZ broth: the above without yeast extract.

L-broth: 10 gm tryptone (Difco); 5 gm yeast extract (Difco); 10 gm NaCI; and H2O to 1 liter.

(2) Log phase amoebae grown in shaken suspension with E. coli B/r. The strains employed are DdB, a subclone by Sussman from Raper's NC-4, and DdC, a cycloheximide-resistant derivative of DdB from Sussman via Rich Kessin.

(3) KPM buffer: 2.25 gm KH2PO4; 0.67 gm K211PO4; 0.50 gm MgSO4.7H2O; and H20 to 1000 ml; pH 6.1.

(4) G-418 (Gibco, 3175 Staley Road, Grand Island, New York 14072); 449 y9 of G-418/mg of solid. This is an impure fermentation product that can be further purified. It is used in the present protocol without further purification. Throughout this specification drug concentration is given as pg/ml of solid, rather than g of active drug. Stocks of G-418 were prepared at 10 mg/ml in water or KPM, and stored as 1 ml aliquots at - 1 0'C to - 20"C in glass or plastic tubes. The drug is inactivated after several freeze-thaw cycles. An aliquot can be used 3 or 4 times before loss of activity is noticeable. It is recommended that an aliquot be used only twice.

B. Preparation of Bacteria: The procedure employed is as follows: (1) Inoculate 50 ml of L-broth in a 250 ml flask with a single colony of E. coli B/r. Grow to saturation overnight at 37"C with shaking.

(2) Inoculate each of five 3000 ml Frenbach flasks containing 1 500 ml NZY, NZ, or L-broth with 5.0 ml of the saturated overnight culture of E. coli B/r.

(3) Grow to saturation (or about 14 hours) at 37"C with shaking (300 rpm). Let bacteria settle for 24 hours (or overnight) at 4"C. Decant the supernatants, recover the bacteria by centrifugation (10 K, 5'), resuspend the pellets completely in a total of 1500 ml of KPM, i.e., 250 ml of buffer into each of 6 centrifuge botties containing pellets of bacteria, and centrifuge again at 10 K for 5'. Decant completely and resuspend the bacteria in KPM to A595 = 60. Store at 4"C.

The cells remain usable for at least 4 weeks at 4"C. It is noted that cells that are not sufficiently washed to remove growth medium appear to produce a metabolite that accumulates during storage and is toxic to amoebae.

C. Amplification of Plasmid-pBM 7.6: (1) Grow 5.0 ml overnight culture of E. coli containing pBM 7.6 or the desired vector under penicillin (100 llg/ml) selection in L-broth. Transfer overnight culture to 1.5 liters sterile L-broth and grow under selection to A595 of 0.25 to 0.30 (mid-log). When cells have reached mid-log, add 375 mg chloramphenicol (250 yg/ml). Shake at 37"C overnight. Recover the bacteria by centrifugation (10 K, 5 min). Resuspend the pellet completely in about 250 ml of buffer and centrifuge again at 10 K for 5 minutes. Decant completely and resuspend the bacteria in KPM buffer to an A595 of 10. These bacteria containing amplified plasmid will be used at A595 of 10 for Dictyostelium transformation.

D. Transformation: (1) Inoculate 20 ml of KPM buffer containing E. coli B/r at a concentration of A595 of 10 with Dictyostelium amoebae.

(2) Harvest at 5-6 x 106 amoebae/ml, wash 5 times with cold KPM buffer, each time centrifuge at 1 k rpm for 5 minutes.

(3) Resuspend pellet in 2 ml KPM. buffer. Count cells and dilute them to 5 X 104--1 X 105 amoebae/ml into the bacteria containing chloramphenicol amplified plasmid. Next morning count cells. When the culture approaches 5 X 106 amoebae/ml, dilute it back to 1 X 105 amoebae/ml using the bacteria containing the chloramphenicol amplified plasmid as a food source. This feeding can be carried out for several days, each time adding fresh bacteria containing plasmid as food source. At the conclusion of a transformation, the amoebae were washed 5 times with cold KPM buffer (each time centrifuged at 1 K rpm for 1 5 minutes) and diluted into 10 ml suspensions of E. coli B/r in KPM buffer at an A595 of 1 5 contained in 250 ml flasks.

(4) Selection was done using G-418 at a concentration of 40-50,ug/ml. The culture was maintained in log phase (104 to 5 X 106 amoebae/ml) by dilution into KPM buffer containing G418 (40,ug/ml) and E. coli B/r (A595 = 15). Growth of amoebae was monitored by directly measuring cell number with a hemacytometer.

The control cells that were fed with E. coli B/r during the entire procedure were drug treated and died after growing for one or two days as seen by a falling cell count in these cultures.

(5) Determining Transformation Efficiency. The fraction of treated cells that became trans formed was determined. Transformed cells were diluted serially into a series of 10 ml cultures of KPM containing G-418 (40 ilg/ml) and E. coli B/r (A595 = 15). The cultures were shaken (250 rpm) at 22 C as above and were monitored for growth during the following seven to fifteen days. The smallest number of cells which yielded a transformed culture was noted. Data is given in Table I. Controls were treated identically except that they were fed E. coli B/r.

T A B L E I Determination of Efficiency By Feeding Transformation 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Number of Cells 1x107 5x106 2.5x107 1.2x108 3.9x109 1.6x10 5x10 1.5x1014 1x106 1.5x106 5.6x107 6x108 1.5x1010 4.5x10 1.5x10 3x1014 1x105 1x104 2x104 4x104 9x105 5x107 2.5x109 5x1010 1x104 < 104* 1x104 2x104 5x104 2.5x106 5x107 1x109 1x103 < 104 1x104 2x105 2.5x106 7.5x106 2x108 4x109 1x102 < 104 < 104 < 104 1x104 3x104 1x106 2x107 1x10 < 104 < 104 < 104 < 104 < 104 2x104 6x105 1 < 104 < 104 < 104 < 104 < 104 < 104 < 104 1x107(WT) 2x106 7x105 2x104 < 104 < 104 < 104 < 104 At the conclusion of 6 days feeding with chloramphenicol amplified pBM 7.6 in LA102 E.coli, the Dictyostelium cells were diluted serially into 10 ml cultures of KPM containing G-418 ((40 g/ml). The cultures' viability was monitored during the following 7 days.The smallest number of initial cells which yielded a G-418 resistant culture was noted. Control wild type (WT) cultures fed with E. coli free of plasmid were treated identically.

* < 104 means no cells observed in hemocytometer of 0.100 mm.

The effect of G-418 on the growth of six days feeding experiment with chloramphenical amplified with pBM 7.6 is illustrated in Fig. 3 of the drawing. To obtain the data of Fig. 3 Dictyostelium amoebae, fed for six days with LA102 containing chloramphenicol amplified pBM 7.6 were divided into two aliquots. One portion (non-transformed) was diluted into suspension cultures containing E. coli B/r at A595 = 1 5. The other aliquot was diluted into E. coli B/r (A595 = 15) containing 40 /19 G-418 ml. Cell numbers were monitored with a hemacytometer during subsequent growth at 22"C (250 rpm). The data is as plotted in Fig. 3.

In the process of the present invention the method of plasmid amplification is performed using prior art techniques. One reference disclosing the chloramphenicol amplification of plasmids is Clewell, D. B., "Nature of E. coli Plasmid Replication In The Presence Of Chloramphenicol." J. Bacterial., 110 (1 972), 667-676.

The type of plasmid employed in accordance with the presently disclosed method is not critical. The plasmids as shown in Figs. 1 and 2 can be utilized. It is highly preferable, however, that the plasmid carry the eukaryotic cell DNA such as Dictyostelium DNA as shown in Fig. 1.

This provides a greater efficiency in transformation and a more stable system.

In the present system any bacteria that the eukaryotic cell, such as Dictyostelium, eats can be used as a carrier of the plasmid for feeding. There are only two requirements for the bacteria (a) that the bacteria will be "transformed" with the desired plasm id; and (b) that the bacteria will be genotypically recombinat minus. In this way the bacterial DNA will not interact with the plasmid DNA and will not alter the plasmid DNA. In the preferred embodiment, E. colistrain LA102 which was previously transformed with plasmid pBM 7.6 was used. This is the presently preferred strain.

In the above-preferred embodiment, the marker utilized was G-418 resistance. However, there is no restriction on the type of marker utilized, either selectable or non-selectable, provided that the marker can be hooked to any functional eukaryotic promoter. The plasmid can have a number of genes hooked to the promoter, with all of them being expressed. The limitation is in the strength of the promoter.

As will be apparent from the above preferred embodiment, a high frequency of transformation is achieved by simply adding intact bacteria loaded with plasmids to Dictyostelium amoebae.

The transformation is efficient and reproducible. The transformants are stable. It appears that one reason for the simplicity and efficiency of the method is because the Dictyostelium amoebae which are lower eukaryotes can grow under fermentation conditions long established for bacteria and yeast. Unlike bacteria and yeast, however, Dictyostelium has no cell walis. Thus, recovery of gene products synthesized by Dictyostelium is very easy, for example by detergent lysis, compared to the difficult methods required to lyse bacteria and yeast. It is possible, therefore, to grow many mammalian genes in Dictyostelium to produce mammalian gene products in a high level of production of the human and animal cell proteins for use in medicine and human and animal nutrition.

As will be apparent to one skilled in the art, various modifications can be made within the scope of the aforesaid description. Such modifications being within the ability of one skilled in the art form a part of the present invention and are embraced by the appended claims.

Claims (10)

1. A method of the production of macromolecules by DNA transformation comprising feeding a simple eukaryotic cell with chloramphenicol-induced plasmid in a bacteria carrier, said bacteria having been transformed by said pladmid and being genotypically recombinant minus.

2. A method according to claim 1 in which the eukaryotic cell is Dictyostelium discoideum.

3. A method according to either of claims 1 or 2 in which the plasmid carries Dictyostelium DNA.

4. A method according to any one of claims 1 to 3 wherein the bacteria carrier is E. coli.

5. The method of claim 4 wherein the E. coli is E. coli strain LA102 transformed with plasmid pBM 7.6.

6. A method for the production of macromolecules by DNA transformation comprising feeding Dictyostelium DNA with a plasmid in a bacteria carrier, said bacteria having been transformed by said plasmid and being genotypically recombinant minus, said plasmid present in said bacteria carrier which is fed to said Dictyostelium DNA homologous to the DNA of a recipient organism.

7. A method for the production of macromolecules by DNA transformation, substantially as hereinbefore described and illustrated by reference to Table 1 and the accompaying drawings.

8. Macromolecules when produced by a method according to any one of claims 1 to 7.

9. A method according to either of claims 6 or 7 in which said bacteria is E. cold.

10. A macromolecule when produced by a method according to claim 9.