DK168302B1 - Method of introducing molecules, especially genetic material into plant cells - Google Patents
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- DK168302B1 DK168302B1 DK325189A DK325189A DK168302B1 DK 168302 B1 DK168302 B1 DK 168302B1 DK 325189 A DK325189 A DK 325189A DK 325189 A DK325189 A DK 325189A DK 168302 B1 DK168302 B1 DK 168302B1
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Description
DK 168302 Bl iDK 168302 Bl i
Den foreliggende opfindelse angår en fremgangsmåde til indføring af molekyler, især genetisk materiale, i intakte planteceller.The present invention relates to a method for introducing molecules, especially genetic material, into intact plant cells.
5 Der kendes forskellige metoder til indføring af molekyler, såsom genetisk materiale, f.eks. plasmid-DNA, RNA, virus eller fragmenter deraf, i dyreceller og protoplaster.Various methods for introducing molecules such as genetic material, e.g. plasmid DNA, RNA, viruses or fragments thereof, in animal cells and protoplasts.
Disse kendte metoder omfatter bl.a. kemiske metoder, elektro-10 poration og mikroinjektion. Disse kendte metoder har vist sig at være anvendelige til både transient og stabil transformation. Alle de beskrevne metoder til opnåelse af transient ekspression har også vist sig at være anvendelige til frembringelse af stabile transformationer.These known methods include: chemical methods, electroporation and microinjection. These known methods have been found to be useful for both transient and stable transformation. All of the methods described for obtaining transient expression have also been found to be useful in producing stable transformations.
15 I forbindelse med indføring af genetisk materiale er der et vigtigt lighedspunkt mellem planteprotoplaster og dyreceller, idet begge udelukkende er afgrænset fra omgivelserne af en plasmamembran. I modsætning hertil omfatter intakte plantecel-20 ler udover en plasmamembran også en for højmolekylære forbindelser ugennemtrængelig cellevæg, der består af et tæt netværk af cel 1ulosefibri1 ler, pectin og ofte også lignin.15 In connection with the introduction of genetic material, there is an important similarity between plant protoplasts and animal cells, both bounded exclusively by the environment of a plasma membrane. In contrast, intact plant cells, in addition to a plasma membrane, also include a high molecular weight impervious cell wall consisting of a dense network of cellulose fibers, pectin and often also lignin.
Ligheden mellem planteprotoplaster og dyreceller underbygges 25 af, at de metoder, som kendes til indføring af genetisk materiale i planteprotoplaster, sædvanligvis også kan anvendes på dyreceller.The similarity between plant protoplasts and animal cells is supported by the fact that the methods known for introducing genetic material into plant protoplasts can usually also be applied to animal cells.
Sådanne metoder omfatter: 30Such methods include:
Calciumphosphatudfældning af plasmid-DNA under dannelse af et krystallinsk produkt. Dette produkt kan optages af dyreceller (Graham og van der Eb, Virology, 52, 456-457, 1973) og af planteprotoplaster (Hain et al., Mol. Gen.Calcium phosphate precipitation of plasmid DNA to form a crystalline product. This product can be taken up by animal cells (Graham and van der Eb, Virology, 52, 456-457, 1973) and by plant protoplasts (Hain et al., Mol. Gen.
35 Genet. 199, 161-168, 1985).35 Genet. 199, 161-168, 1985).
Indkapsling af plasmid-DNA med liposom med efterfølgende polyethylenglycolinduceret fusion med planteprotoplaster 2 DK 168302 B1 (Deshayes et al., EMBO 4, 2731-2737, 1985), eller med dyreceller (Feigner et al., Proc. Natl. Acad. Sci. USA, 84, 7413, 1987).Encapsulation of plasmid DNA with liposome with subsequent polyethylene glycol-induced fusion with plant protoplasts 2 Deshayes et al., EMBO 4, 2731-2737, 1985), or with animal cells (Feigner et al., Proc. Natl. Acad. Sci. USA, 84, 7413, 1987).
5 Anvendelse af polyethylenglycol (PEG). Ved denne metode sættes der til pianteprotoplaster og genetisk materiale polyethylenglycol, sædvanligvis 40% polyethylenglycol 6000 (Krens et al., Nature, 296, 72-74, 1982). På tilsvarende måde kan man anvende polyethylenimin eller poly-L-10 ornithin.5 Use of polyethylene glycol (PEG). By this method, polyethylene glycol, usually 40% polyethylene glycol 6000, is added to plant protoplasts and genetic material (Krens et al., Nature, 296, 72-74, 1982). Similarly, polyethyleneimine or poly-L-10 ornithine can be used.
Elektroporation. Her udsættes en suspension af dyrecel ler eller planteprotoplaster for en kortvarig elektrisk impuls med høj feltstyrke i nærværelse af genetisk mate-15 riale (Neuman et al., EMBO J., 1, 841-845, 1982; Fromm et al.. Nature, 319, 791-793, 1986).Electroporation. Here, a suspension of animal cells or plant protoplasts is subjected to a short-lived, high-field electrical impulse in the presence of genetic material (Neuman et al., EMBO J., 1, 841-845, 1982; Fromm et al., Nature, 319, 791-793, 1986).
Mikroinjektion. Her indføres genetisk materiale i planteprotoplaster (Crossway et al., Mol. Gen. Genet., 20, 179, 20 1986) eller i dyreceller (Cappechi, Cell, 22, 479-488, 1980) ved hjælp af en ultratynd mikropipette.Micro injection. Here, genetic material is introduced into plant protoplasts (Crossway et al., Mol. Gen. Genet., 20, 179, 20 1986) or into animal cells (Cappechi, Cell, 22, 479-488, 1980) using an ultra-thin micropipette.
Fra W0 offentiiggørelsesskrift nr. 89/02464 er det kendt at transformere dyreceller med DNA-fragmenter ved at give cel-25 lerne en ultralydsbehandling, der er tilstrækkelig til at traumatisere cellerne uden at slå cellerne ihjel. På grund af de tidligere nævnte ligheder mellem dyreceller og planteprotoplaster, må det anses for nærliggende, at denne metode også vil kunne anvendes til indføring af DNA-fragmenter i plante-30 protoplaster. Da imidlertid ingen af de tidligere nævnte metoder har vist sig at være egnede til indførelse af plasmid-DNA i intakte planteceller, vil fagmanden, der læser W0 offentliggørelsesskriftet, drage den konklusion, at denne metode formentlig også vil kunne anvendes på protoplaster, men han vil 35 næppe komme på den tanke, at metoden vil kunne anvendes på intakte, vanskeligt gennemtrængelige planteceller. Det er således almindeligt accepteret blandt fagfolk, at plantecellers 3 DK 168302 B1 vægge generelt er uigennemtrængelige for store molekyler såsom DNA og proteiner.From WO Publication No. 89/02464, it is known to transform animal cells with DNA fragments by providing the cells with an ultrasound treatment sufficient to traumatize the cells without killing the cells. Due to the aforementioned similarities between animal cells and plant protoplasts, it should be considered obvious that this method will also be applicable to the insertion of DNA fragments into plant protoplasts. However, since none of the aforementioned methods have been found to be suitable for insertion of plasmid DNA into intact plant cells, those skilled in the art will read WO publication disclosure that this method will probably also be applicable to protoplasts, but he will 35 hardly think that the method can be applied to intact, difficult-to-penetrate plant cells. Thus, it is generally accepted by those skilled in the art that the walls of plant cells are generally impervious to large molecules such as DNA and proteins.
For at løse dette problem har man derfor været nødt til først 5 at fjerne plantecellernes cellevægge, sædvanligvis ved enzymatisk hydrolyse, for derefter at kunne indføre genetisk materiale i de derved dannede protoplaster. Dette giver sædvanligvis vanskeligheder, da det normalt er endnu vanskeligere at få protoplaster regenereret til hele planter sammenlignet med 10 anvendelse af intakte planteceller. Der er således et stort behov for at finde en metode, hvormed man kan indføre genetisk materiale direkte ind i intakte planteceller, således at man undgår problemerne med fremstilling af protoplaster og problemerne med regenerering ud fra protoplaster.Therefore, to solve this problem, it was first necessary to remove the cell walls of the plant cells, usually by enzymatic hydrolysis, in order to then introduce genetic material into the protoplasts thus formed. This usually presents difficulties as it is usually even more difficult to have protoplasts regenerated for whole plants compared to the use of intact plant cells. Thus, there is a great need to find a method by which genetic material can be introduced directly into intact plant cells, so as to avoid the problems of producing protoplasts and the problems of regeneration from protoplasts.
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For nyligt er der udviklet en metode til indføring af plasmid-DNA i intakte planteceller. Denne er baseret på, at små partikler beklædt med plasmid-DNA skydes med en høj hastighed ind i de intakte planteceller (Klein et al., Nature, 327, 70-73, 20 1987). Denne metode kræver dog et meget kostbart udstyr, som endvidere kræver betydelig ekspertise for at kunne anvende det på rette måde.Recently, a method for introducing plasmid DNA into intact plant cells has been developed. This is based on small particles coated with plasmid DNA being pushed into the intact plant cells at a high rate (Klein et al., Nature, 327, 70-73, 20 1987). However, this method requires a very expensive equipment which also requires considerable expertise in order to use it properly.
Der er således et behov for en mere enkel og billig metode til 25 indføring af molekyler, især genetisk materiale, i intakte planteceller.Thus, there is a need for a more simple and inexpensive method for introducing molecules, especially genetic material, into intact plant cells.
Det har nu vist sig, at indføring af molekyler, især genetisk materiale, i intakte panteceller kan gennemføres ved mild ul-30 tralydbehandling ved en metode, der kun kræver forholdsvis billigt og let tilgængeligt udstyr og under opnåelse af en god effektivi tet.It has now been found that the introduction of molecules, especially genetic material, into intact pledge cells can be accomplished by mild ultrasound treatment by a method requiring only relatively inexpensive and readily available equipment and achieving good efficiency.
Det er således formålet med den foreliggende opfindelse at an-35 vise en fremgangsmåde til indføring af molekyler, især genetisk materiale, i intakte planteceller på en billig og effektiv måde.It is thus the object of the present invention to provide a method for introducing molecules, especially genetic material, into intact plant cells in an inexpensive and efficient manner.
DK 168302 B1 4DK 168302 B1 4
Dette opnås ved fremgangsmåden ifølge opfindelsen der er ejendommelig ved at et medium, der indeholder plantecellerne og molekylerne, udsættes for ultralydbehandling med en frekvens fra 5 kHz til 10 MHz, en udgangseffekt på op til 300 watt i et tidsrum på op til 10.000 ms.This is achieved by the method according to the invention characterized in that a medium containing the plant cells and molecules is subjected to ultrasonic treatment at a frequency of 5 kHz to 10 MHz, an output power of up to 300 watts for a period of up to 10,000 ms.
Fremgangsmåden ifølge opfindelsen er et nyttigt alternativ til ® de tidligere kendte indføringsmetoder. Med fremgangsmåden er der opnået en ny metode, der kun kræver beskedne omkostninger, og som kan gennemføres på en hurtig og simpel måde. På basis af de allerede gennemførte indføringsforsøg kan man forudse, at fremgangsmåden også vil vise sin overlegenhed ved anvendel-se på mange andre biologiske materialer, hvor de kendte metoder er utilstrækkelige.The method of the invention is a useful alternative to the prior art insertion methods. The method has achieved a new method that requires only a small cost and can be implemented in a quick and simple way. On the basis of the introductory experiments already carried out, it can be foreseen that the method will also show its superiority when applied to many other biological materials where the known methods are insufficient.
Ved fremgangsmåden ifølge opfindelsen underkastes plantecellerne en ultralydbehandling, der er betydeligt mildere end den ultralydbehandling, der benyttes til homogenisering eller lysis af celler. Det er således vigtigt at behandlingen er passende mild, således at en tilstrækkelig andel af plantecellerne bevarer levedygtigheden. For at sikre dette udsætter man plantecellesuspensionen for ultralydbølger i 20 frekvensområdet fra 5 kHz til 10 MHz, især fra 10 til 100 kHz, med en udgangseffekt (dvs. den til det lydgivende organ tilførte elektriske effekt) på op til 300 watt, såsom 5-300 watt, fortrinsvis 30-70 watt, i et tidsrum på op til 10.000 ms, såsom 100-3000 ms, fortrinsvis 400-1000 ms.In the method of the invention, the plant cells are subjected to an ultrasound treatment which is considerably milder than the ultrasound treatment used for the homogenization or lysis of cells. Thus, it is important that the treatment is suitably gentle so that a sufficient proportion of the plant cells maintains viability. To ensure this, the plant cell suspension for ultrasonic waves in the 20 frequency range is exposed from 5 kHz to 10 MHz, in particular from 10 to 100 kHz, with an output power (ie, the electrical power supplied to the sounding device) of up to 300 watts, such as 5 300 watts, preferably 30-70 watts, for a period of up to 10,000 ms, such as 100-3000 ms, preferably 400-1000 ms.
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Som eksempler på molekyler, der kan indføres i planteceller ved fremgangsmåden ifølge opfindelsen, kan nævnes DNA, plas-mid-DNA, RNA, virus, proteiner, lipider, lægemidler, små molekyler, organeller eller fragmenter af sådanne materialer.Examples of molecules that can be introduced into plant cells by the method of the invention include DNA, plasmid DNA, RNA, virus, proteins, lipids, drugs, small molecules, organelles or fragments of such materials.
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Fremgangsmåden ifølge opfindelsen har med fordel været anvendt til indføring af molekyler i planteceller fra sukkerroe og tobak.The method of the invention has advantageously been used for introducing molecules into sugar beet and tobacco plant cells.
35 Et fordelagtigt medium, hvori plantecellerne og molekylerne er indeholdt under ultralydbehandlingen, er en vandig saltopløsning (CPW), som er nærmere beskrevet nedenfor i forbindelse med eksemplerne, indeholdende 21-28% saccharose.An advantageous medium in which the plant cells and molecules are contained during the ultrasound treatment is an aqueous saline solution (CPW), which is further described below in connection with the Examples, containing 21-28% sucrose.
5 DK 168302 B15 DK 168302 B1
Ved indføring af plasmid-DNA ved fremgangsmåden ifølge opfindelsen opnås sarligt gode resultater, hvis mediet indeholder plasmid-DNA i en koncentration på mindst 10 pg/ml.When introducing plasmid DNA into the method of the invention, very good results are obtained if the medium contains plasmid DNA at a concentration of at least 10 µg / ml.
5 Ved fremgangsmåden ifølge opfindelsen kan man med fordel gennemføre den milde ultralydbehandling under anvendelse af et spidst lydgivende organ, som kun neddyppes i den øvre del af mediet.In the method according to the invention, the gentle ultrasound treatment can advantageously be performed using a pointed sounding device which is only immersed in the upper part of the medium.
10 Ved anvendelse af fremgangsmåden ifølge opfindelsen er der påvist indføring af molekyler i tokimbladede planteceller, såsom sukkerroe og tobak. Det må dog formodes, at fremgangsmåden er lige så velegnet til indførelse af molekyler i enkimbladede planteceller.10 Using the method of the invention, introduction of molecules into two-seeded plant cells such as sugar beet and tobacco has been demonstrated. However, it is believed that the method is equally suitable for introducing molecules into monocotyledonous plant cells.
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Fremgangsmåden ifølge opfindelsen kan med fordel gennemføres ved anvendelse af et apparat, der omfatter et lydgivende organ, der i et medium kan udsende ultralyd i et tidsrum på op til 10.000 ms. Et sådant apparat kan med fordel være udformet 20 til at udsende ultralydbølger med en frekvens i området fra 10 til 100 kHz.Advantageously, the method according to the invention can be carried out by using an apparatus comprising a sounding means which can emit ultrasound in a medium for a period of up to 10,000 ms. Such an apparatus may advantageously be designed to emit ultrasonic waves having a frequency in the range of 10 to 100 kHz.
Til løsning af forskelligartede opgaver kan apparatet være udformet således, at den til det lydgivende 25 organ tilførte effekt kan indstilles på en hvilken som helst værdi i området fra 5 til 300 watt og således, at lydbehandlingens varighed er indstillelig i området fra 100 ms til 10.000 ms.For solving various tasks, the apparatus may be designed such that the power supplied to the sounding means can be set to any value in the range of 5 to 300 watts and so that the duration of the sound processing is adjustable in the range of 100 ms to 10,000. ms.
30 Apparatets lydkilde er med fordel udformet således, at den kan neddyppes i et medium, der er placeret i en passende beholder, såsom et Eppendorfrør. En sådan udformning, hvor det lydafgivende organ sidder for enden af en tynd stav, er kendt fra konventionelle ultralydapparater beregnet til nedbrydning el-35 ler lysis af cellemateriale.Advantageously, the sound source of the apparatus is designed to be immersed in a medium placed in a suitable container such as an Eppendor tube. Such a design, in which the sound-emitting member sits at the end of a thin rod, is known from conventional ultrasonic devices intended for degradation or lysis of cell material.
Det har ved forsøg vist sig at sådanne konventionelle ultralydapparater kan anvendes ved fremgangsmåden ifølge opfinde!- 6 DK 168302 B1 sen. Den foreliggende opfindelse er udviklet under anvendelse af et i handelen tilgængeligt apparat med betegnelsen Sonifier B 15, der leveres af Branson, Eagle Road, Danbury, Connecticut, USA, beregnet til lysis af celler. Apparatet kan udsen-5 de ultralyd med en frekvens på 20 kHz. De i nærværende beskri velse og krav angivne effekter i watt er udgangseffekten, sådan som den aflæses på udgangskontrolskalaen (output control). Ved forsøgene var det ultralydgivende organ neddyppet ca. 2-3 mm fra overfladen. Ved orienterende forsøg under anvendelse af 10 et kalorimeter har det vist sig, at der ved den benyttede for søgsopstilling opnås en afsat effekt til væsken, som er på ca.It has been found by experiment that such conventional ultrasonic apparatus can be used in the method of the invention. The present invention has been developed using a commercially available apparatus designated Sonifier B 15 provided by Branson, Eagle Road, Danbury, Connecticut, USA, for cell lysis. The apparatus can emit ultrasound at a frequency of 20 kHz. The watts specified in this specification and claims are watts output as read on the output control scale. In the experiments, the ultrasound was immersed for approx. 2-3 mm from the surface. In orientation experiments using 10 a calorimeter, it has been found that in the search arrangement used, a set effect is obtained for the liquid, which is approx.
5 til 10% af den angivne udgangseffekt. Ved anvendelse af andre apparater vil fagmanden på samme måde som beskrevet i de efterfølgende eksempler kunne bestemme en passende indstil-15 ling, som giver en mild ultralydbehandling med effektiv indføring af molekyler, under opretholdelse af en tilstrækkelig levedygtighed, det vil sige en indstilling, som giver en ultralydbehandlingseffekt svarende til den, der er anvendt i de ifølge eksemplerne foretagne forsøg. Det kan til sammenligning 20 oplyses, at når det nævnte apparat Sonifier B 15 anvendes til lysis eller homogenisering af celler under i øvrigt samme betingelser, indstiller man det sædvanligvis på en udgangseffekt på 80-100 watt og en behandlingstid på 30.000 til 250.000 ns ved behandling af intakte celler.5 to 10% of the specified output power. Using other apparatus, the skilled artisan will, in the same manner as described in the following examples, be able to determine a suitable setting which provides a gentle ultrasound treatment with efficient introduction of molecules, while maintaining a sufficient viability, that is, a setting which gives an ultrasound treatment effect similar to that used in the experiments performed in the examples. By comparison, it can be stated that when said apparatus Sonifier B 15 is used for lysis or homogenization of cells under otherwise the same conditions, it is usually set to an output power of 80-100 watts and a processing time of 30,000 to 250,000 ns in treatment. of intact cells.
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Som tidligere nævnt kendes der flere metoder til indføring af genetisk materiale i planteceller, hvor disse først omdannes til planteprotoplaster, idet der opnås en højere grad af indføring af det genetiske materiale i protoplasterne, da disse 30 ikke har den spærrende cellevæg. Til gengæld er det for proto-plaster af en række plantearter, især enkimbladede planter, vanskeligt at regenerere protoplaster til intakte planter. Selv om der er opnået gunstige resultater med protoplaster af visse plantearter er der andre plantearter, hvor det vil være 35 mere fordelatigt at benytte intakte planteceller, plantekim eller andet morforgent materiale i stedet for protoplaster. Det er ved forsøg påvist, at direkte indføring af genetisk 7 DK 168302 B1 materiale i intakte celler ved mild ultralydbehandling ved fremgangsmåden ifølge opfindelsen er mulig og attraktiv, da der her er tale om en mindre kompliceret metode, der giver mulighed for en drastisk forøgelse af det antal af plantear-5 ter, hvor der med succes kan gennemføres genetisk manipulation.As mentioned earlier, several methods for introducing genetic material into plant cells are known, where they are first converted into plant protoplasts, with a higher degree of introduction of the genetic material into the protoplasts, since these do not have the blocking cell wall. In contrast, for proto-plastics of a variety of plant species, especially single-seeded plants, it is difficult to regenerate protoplasts for intact plants. Although favorable results have been obtained with protoplasts of certain plant species, there are other plant species where it would be more advantageous to use intact plant cells, plant germs or other morphogenic material instead of protoplasts. It has been shown by experiment that direct introduction of genetic material into intact cells by mild ultrasound treatment according to the method of the invention is possible and attractive, since this is a less complicated method which allows for a drastic increase in the number of plant species where genetic manipulation can be successfully carried out.
Som genetisk materiale kommer navnlig DNA eller fragmenter deraf, såsom plasmid-DNA, på tale. Det må imidlertid antages, 10 at fremgangsmåden også er velegnet til indføring af RNA eller fragmenter deraf samt til indføring af virus, f.eks. til patologiske undersøgelser. Ligeledes vil fremgangsmåden ifølge opfindelsen formentlig også kunne anvendes til indføring af proteiner, lipider, lægemidler, små molekyler samt organeller 15 og viruspartikler i celler.In particular, as genetic material, DNA or fragments thereof, such as plasmid DNA, are discussed. However, it should be assumed that the method is also well suited for introducing RNA or fragments thereof and for introducing viruses, e.g. for pathological examinations. Likewise, the process of the invention will probably also be applicable to the introduction of proteins, lipids, drugs, small molecules as well as organelles and virus particles into cells.
Som medium kan der anvendes et hvilket som helst passende konventionelt medium for celler eller protoplaster.As a medium, any suitable conventional medium for cells or protoplasts may be used.
20 Fremgangsmåden ifølge opfindelsen benytter en teknik, hvor der antageligt sker en midlertidig, moderat svækkelse af såvel cellemembran som cellevæg, hvilket i de efterfølgende eksempler er påvist hos intakte planteceller. Således påvises det i eksemplerne, at plasmid-DNA kan trænge ind i ultralydbehand-25 lede planteceller.The method of the invention employs a technique in which there is likely to be a temporary, moderate weakening of both cell membrane and cell wall, as demonstrated in the following examples in intact plant cells. Thus, it is demonstrated in the Examples that plasmid DNA can penetrate ultrasonically treated plant cells.
Omfanget af opfindelsens anvendelighed vil fremgå af den efterfølgende detaljerede beskrivelse.The scope of the applicability of the invention will become apparent from the following detailed description.
30 « 35 8 DK 168302 B130 «35 8 DK 168302 B1
Eksemplerexamples
Generelle forsøgsbetingelser: 5 CellesuspensionskulturGeneral experimental conditions: 5 Cell suspension culture
En cellesuspensionskultur af sukkerroe (Beta vulgaris L.) af genotypen Ml (fra DANISCO A/S, København, Danmark) fremstilles ud fra callus opnået fra kim (embryo). Cellesuspensionen dyr-10 kes i mørke ved 25eC på et roterende rysteapparat. Cellerne opretholdes ved subdyrkning i et medium ifølge Murashige og Skoog (Physiol. Plant. 15, 473-497, 1962) suppleret med 5,7 μΜ indoleddikesyre og 4,4 μΜ benzyladenin.A cell suspension culture of sugar beet (Beta vulgaris L.) of genotype M1 (from DANISCO A / S, Copenhagen, Denmark) is made from callus obtained from germ (embryo). The cell suspension is grown in the dark at 25 ° C on a rotary shaker. The cells are maintained by sub-culture in a medium according to Murashige and Skoog (Physiol. Plant. 15, 473-497, 1962) supplemented with 5.7 μΜ of indole acetic acid and 4.4 μΜ of benzyladenine.
15 CPW15 CPW
CPW er en vandig opløsning af en blanding af uorganiske salte omfattende blandt andet ca. 10 mM Ca++ beskrevet af Frearson et al., (Dev. Biol. 33, 130-137, 1973).CPW is an aqueous solution of a mixture of inorganic salts comprising, inter alia, ca. 10 mM Ca ++ described by Frearson et al. (Dev. Biol. 33, 130-137, 1973).
2020
UltralvdbehandlinoUltralvdbehandlino
En cellesuspension til ultralydbehandling fremstilles ved at udtage cellerne 3-4 dage efter subdyrkning og vaske to gange i 25 CPW 13S (dvs. CPW indeholdende 13% sorbitol) og suspendering til sidst i CPW 13S i forholdet 1 rumfang celler til 4 rumfang CPW 13S. Til denne suspension i 0,35 ml CPW indeholdende 21% saccharose og indeholdende planteceller (500.000/ml) i et Ep-pendorfrør sættes plasmid DNA til en siutkoncentration på 45 30 μg/ml. Som plasmid DNA anvendes et plasmid, der koder for markør-enzymet chloramphenicol-acetyl transferase (CAT). Det anvendte plasmid er plasmidet pCaMVCN, der med kodebetegnelsen 27-4909 leveres af Pharmacia LKB Biotechnology, Uppsala, Sverige.An ultrasonic cell suspension is prepared by sampling the cells 3-4 days after subculture and washing twice in 25 CPW 13S (ie CPW containing 13% sorbitol) and finally suspending in CPW 13S at 1 volume cells to 4 volumes CPW 13S. To this suspension in 0.35 ml of CPW containing 21% sucrose and containing plant cells (500,000 / ml) in an Ep-pendor tube, plasmid DNA is added to a siut concentration of 45 30 µg / ml. As plasmid DNA, a plasmid encoding the marker enzyme chloramphenicol acetyl transferase (CAT) is used. The plasmid used is the plasmid pCaMVCN, supplied with the code designation 27-4909 provided by Pharmacia LKB Biotechnology, Uppsala, Sweden.
Efter hurtig omrystning neddyppes mikrotippen af en Sonifier B 15 (leveret af Branson, Eagle Road, Danbury, Connecticut, USA) 35 DK 168302 Bl 9 i den øvre halvdel af cellesuspensionen (dvs. 2-3 mm fra overfladen). Der tilføres en ultralydimpuls med en frekvens på 20 kHz. Den effekt, der angives i forsøgene bestemmes ud fra udgangskontrolskalaen (output control), hvor en enhed svarer til 5 15 watt elektrisk effekt. Den effektive akustiske effekt er blevet målt til 0,22 watt/cm2 ved 10 watt elektrisk effekt. Behandlingens varighed indstilles ved hjælp af skalaen for procent driftscyklus (procent duty circle), hvor man ved en indstilling på 10% opnår, at en ultralydimpuls varer i 110 ms.After rapid shaking, the micro tip of a Sonifier B 15 (supplied by Branson, Eagle Road, Danbury, Connecticut, USA) is immersed in the upper half of the cell suspension (i.e., 2-3 mm from the surface). An ultrasonic pulse with a frequency of 20 kHz is applied. The power specified in the tests is determined from the output control scale, where a unit corresponds to 5 15 watts of electrical power. The effective acoustic power has been measured at 0.22 watts / cm 2 at 10 watts electrical power. The duration of treatment is set using the percent duty cycle scale, where, at a setting of 10%, an ultrasonic pulse lasts for 110 ms.
1010
Derefter overføres de ultralydbehandlede celler til petriskåle med et MS-medium (Murashige og Skoog, se ovenfor) og der inkuberes ved 23°C i 2 dage. Derefter påvises tilstedeværelsen af CAT-aktivitet i en ekstrakt udvundet fra de behandlede celler 15 ved tilsætning af l4C-mærket chloramphenicol og opvarmning i 6 minutter til 60eC. Efter afkøling tilsættes acetyl-coenzym A til prøven, således at siutkoncentrationen af acetyl-coenzym A bliver 0,71 mM. Indføringen af plasmidet bedømmes ved måling af den procentuelle omdannelse af chloramphenicol (CA). Den 20 anvendte metode er en modifikation af den metode, der er beskrevet af Gorman et al. (Mol. Cell. Biol. 2, 1044-1051, 1982).Then, the ultrasound-treated cells are transferred to Petri dishes with an MS medium (Murashige and Skoog, see above) and incubated at 23 ° C for 2 days. Then, the presence of CAT activity in an extract extracted from the treated cells is detected by the addition of 14C-labeled chloramphenicol and heating for 6 minutes to 60 ° C. After cooling, acetyl coenzyme A is added to the sample so that the suture concentration of acetyl coenzyme A becomes 0.71 mM. The introduction of the plasmid is assessed by measuring the percentage conversion of chloramphenicol (CA). The method used is a modification of the method described by Gorman et al. (Mol. Cell. Biol. 2, 1044-1051, 1982).
Eksempel 1 25 Nærværende eksempel belyser indføring af plasmid-DNA i intakte celler af sukkerroe af genotypen Ml.Example 1 The present example illustrates introduction of plasmid DNA into intact cells of sugar beet of genotype M1.
Suspensionskulturer af sukkerroeceller opretholdes ved sub-30 dyrkning som beskrevet ovenfor og ultralydbehandies, dyrkes og analyseres på den beskrevne måde. Resultaterne fra den målte CAT-aktivitet fremgår af tabel 1.Suspension cultures of sugar beet cells are maintained by subculture as described above and ultrasonically treated, cultured and analyzed in the manner described. The results of the measured CAT activity are shown in Table 1.
35 10 DK 168302 B135 10 DK 168302 B1
Tabel 1 SukkerroeTable 1 Sugar beet
Effekt Tid Omdannelse af CAPower Time Conversion of CA
(watt) (ms) (%) 500 0,03 60 800 0,06 75 800 0,21 90 800 0,17 105 800 0,07 10(watts) (ms) (%) 500 0.03 60 800 0.06 75 800 0.21 90 800 0.17 105 800 0.07 10
Eksempel 2 Nærværende eksempel belyser indføring af plasmid-DNA i intakte celler af tobak.Example 2 The present example illustrates introduction of plasmid DNA into intact tobacco cells.
1515
En cellesuspensionskultur af tobak opretholdes ved subdyrkning på samme måde som beskrevet ovenfor for subdyrkning af celle-suspensionerne af sukkerroe, idet dog dyrkningsmediet består af et medium ifølge Murashige og Skoog (Physiol. Plant. 15, 20 473-497, 1962) suppleret med 0,2 mg/1 2,4-dichlorphenoxy- eddikesyre, 0,1 mg/1 kinetin, 0,9 mg/1 thiaminhydrochlorid og 0,2 g/1 KH2PO4, pH 6,0.A cell suspension culture of tobacco is maintained by sub-culture in the same manner as described above for sub-culture of the cell suspension of sugar beet, however, the culture medium consists of a medium according to Murashige and Skoog (Physiol. Plant. 15, 20 473-497, 1962) supplemented with 0 , 2 mg / l 2,4-dichlorophenoxyacetic acid, 0.1 mg / l kinetin, 0.9 mg / l thiamine hydrochloride and 0.2 g / l KH 2 PO 4, pH 6.0.
Cellerne udtages 3-4 dage efter subdyrkning og vaskes to gange 25 i CPW13S (dvs. CPW indeholdende 13% sorbitol) og suspenderes til sidst i CPW13S i forholdet 1 rumfang celler til 4 rumfang CPW13S. Herfra udtages prøver på 0,35 ml, som hver tilsættes plasmidet pCaMVCN til en siutkoncentration på 100 pg/ml. Derefter underkastes cellerne ultralydbehandling under de i tabel 30 2 angivne betingelser. Efter dyrkning i 2 dage i det oven nævnte medium ekstraheres cellerne, og CAT-aktiviteten måles. Resultaterne ses i tabel 2.The cells are taken 3-4 days after subculture and washed twice 25 in CPW13S (ie CPW containing 13% sorbitol) and finally suspended in CPW13S in 1 volume cells to 4 volumes CPW13S. From this, samples of 0.35 ml are taken, each of which is added to the plasmid pCaMVCN at a concentration of 100 µg / ml. The cells are then subjected to ultrasound treatment under the conditions set out in Table 30 2. After growing for 2 days in the above medium, the cells are extracted and the CAT activity measured. The results are shown in Table 2.
3535
Claims (9)
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DK325189A DK168302B1 (en) | 1989-06-29 | 1989-06-29 | Method of introducing molecules, especially genetic material into plant cells |
| PCT/DK1990/000166 WO1991000358A1 (en) | 1989-06-29 | 1990-06-28 | A method for introducing molecules, particularly genetic material, into plant cells |
| EP19900910596 EP0480971A1 (en) | 1989-06-29 | 1990-06-28 | A method for introducing molecules, particularly genetic material, into plant cells |
| JP51026590A JPH05500304A (en) | 1989-06-29 | 1990-06-28 | A method of introducing molecules, especially genetic material, into plant cells |
| AU60432/90A AU645260B2 (en) | 1989-06-29 | 1990-06-28 | A method for introducing molecules, particularly genetic material, into plant cells |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DK325189A DK168302B1 (en) | 1989-06-29 | 1989-06-29 | Method of introducing molecules, especially genetic material into plant cells |
| DK325189 | 1989-06-29 |
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| DK325189D0 DK325189D0 (en) | 1989-06-29 |
| DK325189A DK325189A (en) | 1990-12-30 |
| DK168302B1 true DK168302B1 (en) | 1994-03-07 |
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| EP (1) | EP0480971A1 (en) |
| JP (1) | JPH05500304A (en) |
| AU (1) | AU645260B2 (en) |
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| WO (1) | WO1991000358A1 (en) |
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| CA2042093C (en) * | 1990-05-09 | 2002-12-24 | Gyula Hadlaczky | Cell line carrying an excess of mammalian centromeres |
| WO1997009427A1 (en) | 1995-09-08 | 1997-03-13 | Genentech, Inc. | Vegf-related protein |
| US6998116B1 (en) | 1996-01-09 | 2006-02-14 | Genentech, Inc. | Apo-2 ligand |
| US6030945A (en) * | 1996-01-09 | 2000-02-29 | Genentech, Inc. | Apo-2 ligand |
| US5693512A (en) * | 1996-03-01 | 1997-12-02 | The Ohio State Research Foundation | Method for transforming plant tissue by sonication |
| US6469144B1 (en) | 1996-04-01 | 2002-10-22 | Genentech, Inc. | Apo-2LI and Apo-3 polypeptides |
| US6025155A (en) * | 1996-04-10 | 2000-02-15 | Chromos Molecular Systems, Inc. | Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes |
| US6077697A (en) * | 1996-04-10 | 2000-06-20 | Chromos Molecular Systems, Inc. | Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes |
| US5851984A (en) * | 1996-08-16 | 1998-12-22 | Genentech, Inc. | Method of enhancing proliferation or differentiation of hematopoietic stem cells using Wnt polypeptides |
| US6159462A (en) * | 1996-08-16 | 2000-12-12 | Genentech, Inc. | Uses of Wnt polypeptides |
| US6462176B1 (en) | 1996-09-23 | 2002-10-08 | Genentech, Inc. | Apo-3 polypeptide |
| US5990281A (en) * | 1996-09-30 | 1999-11-23 | Genentech, Inc. | Vertebrate smoothened proteins |
| WO1998058062A1 (en) | 1997-06-18 | 1998-12-23 | Genentech, Inc. | Apo-2DcR |
| US6342369B1 (en) | 1997-05-15 | 2002-01-29 | Genentech, Inc. | Apo-2-receptor |
| CA2293724C (en) | 1997-06-05 | 2010-02-02 | Xiaodong Wang | Apaf-1, the ced-4 human homolog, an activator of caspase-3 |
| US6342220B1 (en) | 1997-08-25 | 2002-01-29 | Genentech, Inc. | Agonist antibodies |
| ATE411385T1 (en) | 1998-01-15 | 2008-10-15 | Genentech Inc | APO-2 LIGAND |
| US6114603A (en) * | 1998-03-27 | 2000-09-05 | John Innes Center | Genetic engineering of sugarbeet plants |
| DE19834612A1 (en) * | 1998-07-31 | 2000-02-24 | Dornier Medtech Holding Int Gmbh | Method for intracellular transfer of oligonucleotides and device for carrying out the same |
| US6195936B1 (en) * | 1999-02-22 | 2001-03-06 | University Of Iowa Research Foundation | Method for uptake of a substance into a seed |
| JP5118796B2 (en) | 1999-06-28 | 2013-01-16 | ジェネンテック， インコーポレイテッド | Method for producing Apo-2 ligand using divalent metal ions |
| DE19962904A1 (en) * | 1999-12-23 | 2001-08-09 | Dornier Medizintechnik | Apparatus continuously introducing molecules into cells, avoiding their total disruption, focuses controlled acoustic pulses at axis of duct passing carrier fluid |
| ATE511857T1 (en) | 2000-02-16 | 2011-06-15 | Genentech Inc | ANTI-APRIL MONOCLONAL ANTIBODIES AND THEIR USE IN THE TREATMENT OF IMMUNE DISEASES OR CANCER |
| EP2390256A1 (en) | 2001-05-30 | 2011-11-30 | Agrisoma, Inc. | Plant artificial chromosomes, uses thereof and methods of preparing plant artificial chromosomes |
| CN1575338B (en) | 2001-08-29 | 2012-05-16 | 杰南技术公司 | Bv8 nucleic acids and polypeptides having mitogenic activity |
| JP2005508162A (en) | 2001-10-02 | 2005-03-31 | ジェネンテック・インコーポレーテッド | Apo-2 ligand variants and methods of use |
| EP2332531B1 (en) | 2001-11-13 | 2019-07-10 | Genentech, Inc. | Methods of purifying Apo2-Ligand/TRAIL |
| DE10223196B4 (en) | 2002-05-24 | 2004-05-13 | Dornier Medtech Systems Gmbh | Method and device for transferring molecules into cells |
| CA2489348A1 (en) | 2002-06-24 | 2003-12-31 | Genentech, Inc. | Apo-2 ligand/trail variants and uses thereof |
| CA2515288A1 (en) | 2003-03-12 | 2004-09-23 | Genentech, Inc. | Compositions with hematopoietic and immune activity |
| WO2005000351A2 (en) | 2003-06-05 | 2005-01-06 | Genentech, Inc. | Combination therapy for b cell disorders |
| DE602005019367D1 (en) | 2004-12-15 | 2010-04-01 | Dornier Medtech Systems Gmbh | Enhanced cell therapy and tissue regeneration via shock waves in patients with cardiovascular and neurological diseases |
| GB2452543B (en) * | 2007-09-07 | 2012-07-25 | Wei Huang | Nucleic acid transfer techniques |
| DK2233149T3 (en) | 2007-10-16 | 2016-05-17 | Zymogenetics Inc | COMBINATION OF TRANSMEMBRANAKTIVATOR AND CALCIUM MODULATOR AND cyclophilin-LIGAND INTERAKTOR (TACI) AND ANTI-CD20 MEANS FOR TREATMENT OF AUTO-IMMUNE DISEASE |
| US8669085B2 (en) | 2009-02-05 | 2014-03-11 | Ut-Battelle, Llc | Transformation of gram positive bacteria by sonoporation |
| IL264336B2 (en) | 2009-08-11 | 2024-02-01 | Genentech Inc | Production of proteins in glutamine-free cell culture media |
| JP2014513128A (en) | 2011-05-03 | 2014-05-29 | ジェネンテック， インコーポレイテッド | Vascular disruptors and their use |
| KR101791563B1 (en) * | 2015-05-11 | 2017-11-02 | 대한민국 | Method for delaying the ripening of fruits using sound treatment |
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| DE3537261A1 (en) * | 1985-10-19 | 1987-04-30 | Gca Corp | METHOD AND MEDIUM FOR FIELD-INDUCED INSERTING MACROMOLECULES IN LIVING CELLS |
| ES2061720T3 (en) * | 1987-05-05 | 1994-12-16 | Sandoz Ag | TRANSFORMATION OF PLANT TISSUE. |
| GB8721015D0 (en) * | 1987-09-07 | 1987-10-14 | Amersham Int Plc | Modifying living cells |
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1990
- 1990-06-28 AU AU60432/90A patent/AU645260B2/en not_active Expired - Fee Related
- 1990-06-28 WO PCT/DK1990/000166 patent/WO1991000358A1/en not_active Application Discontinuation
- 1990-06-28 JP JP51026590A patent/JPH05500304A/en active Pending
- 1990-06-28 EP EP19900910596 patent/EP0480971A1/en not_active Ceased
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| AU645260B2 (en) | 1994-01-13 |
| AU6043290A (en) | 1991-01-17 |
| EP0480971A1 (en) | 1992-04-22 |
| DK325189D0 (en) | 1989-06-29 |
| JPH05500304A (en) | 1993-01-28 |
| DK325189A (en) | 1990-12-30 |
| WO1991000358A1 (en) | 1991-01-10 |
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