CA1208146A - Method of transferring genes into cells - Google Patents
Method of transferring genes into cellsInfo
- Publication number
- CA1208146A CA1208146A CA000439852A CA439852A CA1208146A CA 1208146 A CA1208146 A CA 1208146A CA 000439852 A CA000439852 A CA 000439852A CA 439852 A CA439852 A CA 439852A CA 1208146 A CA1208146 A CA 1208146A
- Authority
- CA
- Canada
- Prior art keywords
- cells
- genes
- gene
- transferred
- mixture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
A method is provided for transferring genes into cells which comprises the step of subjecting the mixture of genes to be transferred and the target cells to an electric treatment.
A method is provided for transferring genes into cells which comprises the step of subjecting the mixture of genes to be transferred and the target cells to an electric treatment.
Description
`. " ~Z~ 6 A-37344/AJT/RJS/l . METHOD OF TRANSFERRING GENES INTO CELLS
.- The present invention is a meth~d t~ transfer genes to procaryotic or eucaryotic sells. In the fields of genetic engineering, cell biology; and embrys manipulation, various chemical and mechanical methods have been developed for transferring genetic materials into cells. Chemical methods involve the use of chemicals which permeabilize the cell surface, hence facilitates the transf~r of the genetic materials into cells~ [For reviews, see: Gerard Venema ~Bacterial Transformation" in Ad~. Microbiol. Physl. (1979) _: 245-331; George Scangos ~nd Frank H Ruddle "Mechanisms and Applications of DNA-mediated Gene Transfer in M~mmalian Cells - A Review" in Gene (1981) 14: 1-10; O. Wesley McBride and Jane L. Peterson "Chromosome-mediated Gene Transfer in Mammalian Cells" in Ann. Rev. Genet. (1980) 14: 321-345;
Jurgen Horst et al., "On Procaryotic Gene Expression in Eucaryotic Systems" in Human Genetics (198G) 54: 28q-302;
R. Fraley and D. Papahadjopoulos, "New Generation Li~osomes:
The Engineering of an Pfficient Vehicle for Intracellular 20 Delivery of Nucleic Acids" in Trends Biochem. Sci.
(1981) March. pp. 77-80.~ Mechanical methods involve the injection of genetic materials directly into the cells, commonly known as microinjection ~For review, see: W.
French Anderson and Elaine G. Diacumakos "~enetic Engineering in Mammalian Cells" in Scientific American tl981) 245:
106-121).
In procaryotic systems, the chemical methods of ~ransferring genes are usually employed, whereas in eucaryotic systems, both the chemical and mechanical methods are used.
~ 2G~
~2--All methods available at the present moment, however, are somewhat dependent upon hoth the gene which is ~o be transferred and the recipient cells. Methods which may be used to transfer genes into procaryotes may not work in transferring genes into eucaryotes. How~ver, ~ccording to - the present invention, a single method is provided which may be utilized to transfer genes into either procaryotic or eucaryotic cells.
Tigure 1 is a diagram of the apparatus employed in the preferred embodiment of the inv~ntion.
Fiyure 2 is a diagram o~ the apparatus employed in a second embodiment of the invention Figure 3 is an enlarged view of the preferred receptacle shown in Figure 1.
According to the present invention a solution, suspension or other mixture containing the gene to be transferred and the target cell5 are placed in a receptacle such ~hat one electrode contacts the solution, suspension or mixture bel~w the surface thereof. Preferably ~he said elec~rode is located at the lowest point of the said receptacle. Juxtaposed above the surface of the solution, suspension or mixture, but not in contact therewith is a second electrode directed towards the surface of the solution, suspension or mixture~
The distance between the point of the second electrode and the surface of the solution, ~uspension or mixture is not critical. A distance of about 0.7 cm to about 4 cm has been used.
The electrodes may be connected to a conventional electric field generator. The electric field which may be applied to the solution or ~uspension containing the cells and gene must be high enouyh to create a high electric ~ield or electric discharge but not great enough t~ substantially alter or destroy the cells or the gene. Voltages fr~m approximately 3 kilovolts to 20 kilovolts may be used.
When only electric field is applied to the gene~cells mixture, the duration of the fiel~ varies from ~bou~ 1 ~o 90 seconds depending on the nature of the target cells.
When discharge condition is employed, a pulse di~charge up to about 1 second is preferred. The number of pulses which may be applied to the solution, suspension or mixture containing the cells or gene may vary from about several pulses to about 300 pulses, depending upon the pulse width and intensity and the nature of the cells.
Referring to Figure 1, there is shown a conventional electric field generator 10 having a control and monitoring means 11 for applying field or pulse and an intensity control and measuring means 12 for voltage. Such a conventional generator is available~ for example, from Andy Hish Associates, Van Nuys, Model Number ESD255 Electrostatic Discharge Generator with probe P255-1, Probe 13 is connected to said generator 10 ~nd vertically disposed above vial receptacle 15. At the lowest point of vial receptacle 15 is located a ground electrode 14, ~ial receptacle lS
contains a solution or suspension of cells and genes. As shown, vial receptacle 15 may be conical in shape and the ground electrode 14 is located at the apex thereof. ~ithout limiting the invention to any particular theory, the shape of vial receptacle 15 may be preferred since there may be a concentration gradient of cells wi~hin the solution ox suspension 16 due to the heterogen~ity of the cells and in such case the gradient concentration o~ cells may be near the apex of vial receptacle 15.
Referring to ~igure 2 there is shown a second embodiment of the invention. Figure 2 is similar to Figure 1 except that receptacle 17 is a tube with a round bot~om having the ground electrode located at the lowest point of the tube.
ReferringtoFigure 3 there is ~hown ~n enlarged view of vial receptacle 15. As 8hown, a probe 13 is directed towards the surface 18 of the 801ution or ~uspension containing the cells and genes. Both the probe 13 and the vial 15 containing solution 16 may be exposed to the a ~osphere durin~ thP experiment. It is readily apparent that ~he vial 15 need not be completely filled as 6hown in order to per-form the expeximent. A requirement is that the cells and gene containing solution or suspension is placed between direct discharge or electric field from probe 13 to qround electrode 14.
While not limiting the present invention to a pArticular theory, it is believed that by exposing the cells and genes to a high intensity electric field or electric discharge in the above described manner alters the cell surface sufficiently to allow the passage of the gene therethrough.
According to the present invention genes may be transferred to eucaryotic cells. For example, Herpes simplex viru~
thymidine kinase gene may be transferred into mouse L~. (TX ) cells.l Also the E. coli xanthine guanine-phosphoribosyltrans-ferase gene (Ecogpt)2 alone or toghethex with the Herpes simplex ~irus thymidine kinase gene may be transferred into human ClO~HGPRT )3 or C10 lHGPRT TK ) cells, respectively.
Even the bacterial mercury resistance gene which is in the plas~id pKT004 may b~ transferred in~o mouse LM (TK ) cells and renders the mouse cells mercury resistant, In all cases, cells which received the corresponding genes grow under conditions in which the parental cells do not survive.
According to the present invention genes may also be trans-ferred into procaryotic cells. For example, various plasmids shown below in ~able 1 may be transferred i~to E. coli of strains HB101,5 ~Rl,5 or M157.
~8~6 PLASMIDS TRA~SFERRED INTO E. coli CELLS
Plasmids Genetic Marker(s) pBR322(5) AmpR~ TetR
5 pACY~184(6) ~etR, camR
pAcycl77(6) AmpR KanR
pTWS(B) Lac pXTo04(9) ~gR
pAGO(10) AmpR
(X) Abbreviation: ~mp: ~mpicillin; Tet; Tetracycline;
Cam: Chloramphenicol; Kan: ~anamycin;
R: Resistance; Lac~: Lactose pos~tive Hg: Mercury According to the present invention virtually any gene may be 1~ transferred into virtually any target cells, as exemplified in, ~ut not limited to, the followin~
mercury resistance gene into plant cells (carrot, tomato, tobacco, barley, etc.) plasmids/genes into yeast;
plasmids/genes into Streptomyces;
plasmids/genes into Bacillus;
plasmid Ti alone or together with other genes into plant cells;
plasmids/genes into animal embryos or fertilîzed eggs or oocytes;
plasmids/genes into Pseudomonas; etc.
~he aenes which may be transferred according to the present invention may be structur~ genes, i.e., DN~ ~ragments com-prising the gene, or m~y be genes on vectors, such as, plasmid vectors, bacteriophage vectors, viral vectors or yeast vectorsO
~or purposes of illustrating the present in~ention the follow-ing examples are providea. However, ~he sc~pe o~ the invention is not intended to be limited thereto.
-6~ 6 EXAMPLE 1: Gene Transfer in Euca~yotic System The preparation of cells for gene transfer is described as follows. Mouse LM (TR ~ cells were grown in 75 cm2 cell culture flasks with RPMI 1640 mediumll ~upplemen~ed with 10% fetal calf serum at 37 C in a humidified atmosphere containing 5% carbon dioxide to a cell number of around lx107 cell~. Cells were washed, ~rypsinized, pelle~ed hy centrifugation, and ~hen resuspended in phosphate buffered saline (PBS).12 ~
A typical 150 ~1 gene-cells mixture consists of 5xlO to 6X106 cells, 1 ~g or less plasmid pAGO DNA, and 30 ~g or less sheared calf thymus DNA as carrier. The whole mixture was pipetted into the receptacle and subjected to the ~lectric field utilzing the apparatus as 6hown in Figure 1. The applied voltage was between 3.0 kilovolts to 20 kilovolts.
The numher of discharges is 75 at the frequen~y of about 1 discharge per second. At the end of the aischarge treat-ment, the gene-cells mixture was pipetted into cell culture flasks (75 ~m ) each containing 10 ml of R~MI 1640 medium supplemented with 10% fetal calf serum. After 24 hours of incubation at 37 C, 10 ml medium containing 2xHAT13 were added. After around one to two weeks of incubation, cells which received the plasmid p~GO DNA survived as thymidine kinase positive clones. ~hose cells which did not receive ~5 the gene died.
When the DNA fragment ~3.4 kilobases in size) harbouring the thymidine kinase gene ~n lieu of the whole plasmid pAGO was used, thymidine kinase positive clones were also obtained. In addition, thymidine kinase positive clones were obtained when an electric field was used, in lieu of the said discharge.
~2~ 6 EXAMPLE 2: Gene Transfer in procaryotic system.
Bacteria E. coli strain HB101 were yrown in 10 ml of Luria broth (10 g/l tryptone, 5 g/l yeast ext~act, 5 g/l NaCl) at 37C to a cell density of about 6xlOB ~ells/ml.
Between 10 to 50 ng of plasmid pBR322 DNA were mixed with 100 ~1 of the bacteria. Plasmid pBR322 harbors two selectable genes, namely, ampicillin and tetracycline resistance genes. The gene-cells mixture was subjected to electrical treatment util-zing -the apparatus shown in Figure 1 and as described in Example 1. Trans~ormants were selected by plating bacteria-plasmid mixture onto agar plates [1.35~ (w/v) Difco agar in Luria bro~h]
supplemented with either ampicillin (30 ~g/ml~ or tetracycline (15 ~g/ml). Agar plates were incubated at 37C for at least 14 hours. Both ampicillin and tetra-cycline resistance bacterial colonies were obtained.
Experience has shown that when an "old" bacterial culture, i.e., an overnight culture which was left at room temperature for at least 24 hours, was used for ~he experiment carried out as described above, transformants were also obtained.
Experience has shown also that when E. coli strain M15 was used as the recipient cells for the plasmid pTW5 DNA using the lactose positive colonies selection system 14, lactose positive colonies were obtained.
8~
S. ~it, D. Dubb~, L. Piekarski, T, ~su~ DeletiOn of Thymidine ~inase Acti~ity ~rom L Cells Resistant ~o Bromodeo~yuridine, Exptl. Cell Research 31 (1963~ pp 2gl-312.
R.C. Mulligan, ~. Berg, Expression of a ~a~teri*l Gene in Mam~alian Cells, Science, 209 ~19B0~, pp. 1422-1427 3W. ~erthold, C. Tan, Y.H. Tan, Purification and in vitro Labelling of Interferon Fr~m a ~uman ~ibroblastoid Cell ~ine, J. ~iol. Chem./ 253 (1978) pp. 5206 5212; ~nd unpublished re~ult.
4R. Haars, Y.~. Tan~ unpublished r~sult 5R.L. Rodriguez, R. Tait, ~. Shine, F. ~olivar, H. ~eyneker, M. Betlach, H,W. Boyer, Characterization of Tetracycline and Ampicillin Resistant Plasmid Cloning Vehicles, in Molecular Cloning of Rec~mbinant DNA. Ed. ~y W.A. Scott and R. Werner.
Academic Press (1977) pp. 73-84, 6A.C.Y. Ch ng, S.N. Cohenr Construction and Characterization of Amplifi~ble Multicopy DNA Cl~ning Vehicles Derived from the pl5A Cryptic Plasmid, ~. Bact., 134 (1978) pp. 1141-1156.
7J,R. Beckwith, A Deletion ~naylsis of the ~ac Operator Region in Escherichia coli, ~, Mol. BiDl,~ B ~1964) pp 427-430.
Tai-kin Wong, Attempts to Characterize the aLate~Template"
Properties of T5 DNA after Infection of E, ~bli and Some Aspects on the Mechanism of Plasmid pSC101 M~ ated Te~ra-eycline R~sistance, Ph.D. Dissertation, Ruprecht-Xarl-Universitat Heidelberg, lg78. West Germany.
9~.N. Timmis, F, Cabello, S,N, Cohen, Cloning and Characterization of EcoRI and HinaIII Restriction Endonuclease-generated Fragments of Antibiotic Resistanc~ Plasmi~s R6 5 and R6, ~olec. Gen.
Genet. 162 ~1978) pp. 121-137.
10E, Colbere-Garapin, S. Chousterman, ~, Horodniceanu, ~.
Kourilsky, ~.C. Garapin, Cloning of the Ac~ive Thymidine Xinase Gene of Herpes Sl~mplex Virus ~ype 1 in Escherichia coli, Proc Natl. Acad. Sci. ~SA, 76 tlg79) pp. 3755~3759.
llGibco~ Grand I~land Biological Company, Catalog No, 430-1800.
~9 i~2~
oxoid Limited, En~land~ Code BR14a, 13RPMI 1640 Supplemented with Hypoxanthine 13.6 ~g/ml; .
Aminopterin 0.176 ~g/ml; thymidine 3,78 yg/mlt modified from E.B. 5zybalska, W. Szybalski, Genetics of ~uman Cell Lines. IV. DNA-mediated Heritable Transform~tion of a Bioch~mical Trait, Proc. Natl. Acad. Sci. USA, 48: (1962) pp. 2026-2034, MacConkey Agar Supplemented with 20mM IP~G, ~Isopropyl-~-D-thiogalactopyranoside~ Difco, Catalog No~ 00~5-05, IPTG: Sigma 1-5502.
.- The present invention is a meth~d t~ transfer genes to procaryotic or eucaryotic sells. In the fields of genetic engineering, cell biology; and embrys manipulation, various chemical and mechanical methods have been developed for transferring genetic materials into cells. Chemical methods involve the use of chemicals which permeabilize the cell surface, hence facilitates the transf~r of the genetic materials into cells~ [For reviews, see: Gerard Venema ~Bacterial Transformation" in Ad~. Microbiol. Physl. (1979) _: 245-331; George Scangos ~nd Frank H Ruddle "Mechanisms and Applications of DNA-mediated Gene Transfer in M~mmalian Cells - A Review" in Gene (1981) 14: 1-10; O. Wesley McBride and Jane L. Peterson "Chromosome-mediated Gene Transfer in Mammalian Cells" in Ann. Rev. Genet. (1980) 14: 321-345;
Jurgen Horst et al., "On Procaryotic Gene Expression in Eucaryotic Systems" in Human Genetics (198G) 54: 28q-302;
R. Fraley and D. Papahadjopoulos, "New Generation Li~osomes:
The Engineering of an Pfficient Vehicle for Intracellular 20 Delivery of Nucleic Acids" in Trends Biochem. Sci.
(1981) March. pp. 77-80.~ Mechanical methods involve the injection of genetic materials directly into the cells, commonly known as microinjection ~For review, see: W.
French Anderson and Elaine G. Diacumakos "~enetic Engineering in Mammalian Cells" in Scientific American tl981) 245:
106-121).
In procaryotic systems, the chemical methods of ~ransferring genes are usually employed, whereas in eucaryotic systems, both the chemical and mechanical methods are used.
~ 2G~
~2--All methods available at the present moment, however, are somewhat dependent upon hoth the gene which is ~o be transferred and the recipient cells. Methods which may be used to transfer genes into procaryotes may not work in transferring genes into eucaryotes. How~ver, ~ccording to - the present invention, a single method is provided which may be utilized to transfer genes into either procaryotic or eucaryotic cells.
Tigure 1 is a diagram of the apparatus employed in the preferred embodiment of the inv~ntion.
Fiyure 2 is a diagram o~ the apparatus employed in a second embodiment of the invention Figure 3 is an enlarged view of the preferred receptacle shown in Figure 1.
According to the present invention a solution, suspension or other mixture containing the gene to be transferred and the target cell5 are placed in a receptacle such ~hat one electrode contacts the solution, suspension or mixture bel~w the surface thereof. Preferably ~he said elec~rode is located at the lowest point of the said receptacle. Juxtaposed above the surface of the solution, suspension or mixture, but not in contact therewith is a second electrode directed towards the surface of the solution, suspension or mixture~
The distance between the point of the second electrode and the surface of the solution, ~uspension or mixture is not critical. A distance of about 0.7 cm to about 4 cm has been used.
The electrodes may be connected to a conventional electric field generator. The electric field which may be applied to the solution or ~uspension containing the cells and gene must be high enouyh to create a high electric ~ield or electric discharge but not great enough t~ substantially alter or destroy the cells or the gene. Voltages fr~m approximately 3 kilovolts to 20 kilovolts may be used.
When only electric field is applied to the gene~cells mixture, the duration of the fiel~ varies from ~bou~ 1 ~o 90 seconds depending on the nature of the target cells.
When discharge condition is employed, a pulse di~charge up to about 1 second is preferred. The number of pulses which may be applied to the solution, suspension or mixture containing the cells or gene may vary from about several pulses to about 300 pulses, depending upon the pulse width and intensity and the nature of the cells.
Referring to Figure 1, there is shown a conventional electric field generator 10 having a control and monitoring means 11 for applying field or pulse and an intensity control and measuring means 12 for voltage. Such a conventional generator is available~ for example, from Andy Hish Associates, Van Nuys, Model Number ESD255 Electrostatic Discharge Generator with probe P255-1, Probe 13 is connected to said generator 10 ~nd vertically disposed above vial receptacle 15. At the lowest point of vial receptacle 15 is located a ground electrode 14, ~ial receptacle lS
contains a solution or suspension of cells and genes. As shown, vial receptacle 15 may be conical in shape and the ground electrode 14 is located at the apex thereof. ~ithout limiting the invention to any particular theory, the shape of vial receptacle 15 may be preferred since there may be a concentration gradient of cells wi~hin the solution ox suspension 16 due to the heterogen~ity of the cells and in such case the gradient concentration o~ cells may be near the apex of vial receptacle 15.
Referring to ~igure 2 there is shown a second embodiment of the invention. Figure 2 is similar to Figure 1 except that receptacle 17 is a tube with a round bot~om having the ground electrode located at the lowest point of the tube.
ReferringtoFigure 3 there is ~hown ~n enlarged view of vial receptacle 15. As 8hown, a probe 13 is directed towards the surface 18 of the 801ution or ~uspension containing the cells and genes. Both the probe 13 and the vial 15 containing solution 16 may be exposed to the a ~osphere durin~ thP experiment. It is readily apparent that ~he vial 15 need not be completely filled as 6hown in order to per-form the expeximent. A requirement is that the cells and gene containing solution or suspension is placed between direct discharge or electric field from probe 13 to qround electrode 14.
While not limiting the present invention to a pArticular theory, it is believed that by exposing the cells and genes to a high intensity electric field or electric discharge in the above described manner alters the cell surface sufficiently to allow the passage of the gene therethrough.
According to the present invention genes may be transferred to eucaryotic cells. For example, Herpes simplex viru~
thymidine kinase gene may be transferred into mouse L~. (TX ) cells.l Also the E. coli xanthine guanine-phosphoribosyltrans-ferase gene (Ecogpt)2 alone or toghethex with the Herpes simplex ~irus thymidine kinase gene may be transferred into human ClO~HGPRT )3 or C10 lHGPRT TK ) cells, respectively.
Even the bacterial mercury resistance gene which is in the plas~id pKT004 may b~ transferred in~o mouse LM (TK ) cells and renders the mouse cells mercury resistant, In all cases, cells which received the corresponding genes grow under conditions in which the parental cells do not survive.
According to the present invention genes may also be trans-ferred into procaryotic cells. For example, various plasmids shown below in ~able 1 may be transferred i~to E. coli of strains HB101,5 ~Rl,5 or M157.
~8~6 PLASMIDS TRA~SFERRED INTO E. coli CELLS
Plasmids Genetic Marker(s) pBR322(5) AmpR~ TetR
5 pACY~184(6) ~etR, camR
pAcycl77(6) AmpR KanR
pTWS(B) Lac pXTo04(9) ~gR
pAGO(10) AmpR
(X) Abbreviation: ~mp: ~mpicillin; Tet; Tetracycline;
Cam: Chloramphenicol; Kan: ~anamycin;
R: Resistance; Lac~: Lactose pos~tive Hg: Mercury According to the present invention virtually any gene may be 1~ transferred into virtually any target cells, as exemplified in, ~ut not limited to, the followin~
mercury resistance gene into plant cells (carrot, tomato, tobacco, barley, etc.) plasmids/genes into yeast;
plasmids/genes into Streptomyces;
plasmids/genes into Bacillus;
plasmid Ti alone or together with other genes into plant cells;
plasmids/genes into animal embryos or fertilîzed eggs or oocytes;
plasmids/genes into Pseudomonas; etc.
~he aenes which may be transferred according to the present invention may be structur~ genes, i.e., DN~ ~ragments com-prising the gene, or m~y be genes on vectors, such as, plasmid vectors, bacteriophage vectors, viral vectors or yeast vectorsO
~or purposes of illustrating the present in~ention the follow-ing examples are providea. However, ~he sc~pe o~ the invention is not intended to be limited thereto.
-6~ 6 EXAMPLE 1: Gene Transfer in Euca~yotic System The preparation of cells for gene transfer is described as follows. Mouse LM (TR ~ cells were grown in 75 cm2 cell culture flasks with RPMI 1640 mediumll ~upplemen~ed with 10% fetal calf serum at 37 C in a humidified atmosphere containing 5% carbon dioxide to a cell number of around lx107 cell~. Cells were washed, ~rypsinized, pelle~ed hy centrifugation, and ~hen resuspended in phosphate buffered saline (PBS).12 ~
A typical 150 ~1 gene-cells mixture consists of 5xlO to 6X106 cells, 1 ~g or less plasmid pAGO DNA, and 30 ~g or less sheared calf thymus DNA as carrier. The whole mixture was pipetted into the receptacle and subjected to the ~lectric field utilzing the apparatus as 6hown in Figure 1. The applied voltage was between 3.0 kilovolts to 20 kilovolts.
The numher of discharges is 75 at the frequen~y of about 1 discharge per second. At the end of the aischarge treat-ment, the gene-cells mixture was pipetted into cell culture flasks (75 ~m ) each containing 10 ml of R~MI 1640 medium supplemented with 10% fetal calf serum. After 24 hours of incubation at 37 C, 10 ml medium containing 2xHAT13 were added. After around one to two weeks of incubation, cells which received the plasmid p~GO DNA survived as thymidine kinase positive clones. ~hose cells which did not receive ~5 the gene died.
When the DNA fragment ~3.4 kilobases in size) harbouring the thymidine kinase gene ~n lieu of the whole plasmid pAGO was used, thymidine kinase positive clones were also obtained. In addition, thymidine kinase positive clones were obtained when an electric field was used, in lieu of the said discharge.
~2~ 6 EXAMPLE 2: Gene Transfer in procaryotic system.
Bacteria E. coli strain HB101 were yrown in 10 ml of Luria broth (10 g/l tryptone, 5 g/l yeast ext~act, 5 g/l NaCl) at 37C to a cell density of about 6xlOB ~ells/ml.
Between 10 to 50 ng of plasmid pBR322 DNA were mixed with 100 ~1 of the bacteria. Plasmid pBR322 harbors two selectable genes, namely, ampicillin and tetracycline resistance genes. The gene-cells mixture was subjected to electrical treatment util-zing -the apparatus shown in Figure 1 and as described in Example 1. Trans~ormants were selected by plating bacteria-plasmid mixture onto agar plates [1.35~ (w/v) Difco agar in Luria bro~h]
supplemented with either ampicillin (30 ~g/ml~ or tetracycline (15 ~g/ml). Agar plates were incubated at 37C for at least 14 hours. Both ampicillin and tetra-cycline resistance bacterial colonies were obtained.
Experience has shown that when an "old" bacterial culture, i.e., an overnight culture which was left at room temperature for at least 24 hours, was used for ~he experiment carried out as described above, transformants were also obtained.
Experience has shown also that when E. coli strain M15 was used as the recipient cells for the plasmid pTW5 DNA using the lactose positive colonies selection system 14, lactose positive colonies were obtained.
8~
S. ~it, D. Dubb~, L. Piekarski, T, ~su~ DeletiOn of Thymidine ~inase Acti~ity ~rom L Cells Resistant ~o Bromodeo~yuridine, Exptl. Cell Research 31 (1963~ pp 2gl-312.
R.C. Mulligan, ~. Berg, Expression of a ~a~teri*l Gene in Mam~alian Cells, Science, 209 ~19B0~, pp. 1422-1427 3W. ~erthold, C. Tan, Y.H. Tan, Purification and in vitro Labelling of Interferon Fr~m a ~uman ~ibroblastoid Cell ~ine, J. ~iol. Chem./ 253 (1978) pp. 5206 5212; ~nd unpublished re~ult.
4R. Haars, Y.~. Tan~ unpublished r~sult 5R.L. Rodriguez, R. Tait, ~. Shine, F. ~olivar, H. ~eyneker, M. Betlach, H,W. Boyer, Characterization of Tetracycline and Ampicillin Resistant Plasmid Cloning Vehicles, in Molecular Cloning of Rec~mbinant DNA. Ed. ~y W.A. Scott and R. Werner.
Academic Press (1977) pp. 73-84, 6A.C.Y. Ch ng, S.N. Cohenr Construction and Characterization of Amplifi~ble Multicopy DNA Cl~ning Vehicles Derived from the pl5A Cryptic Plasmid, ~. Bact., 134 (1978) pp. 1141-1156.
7J,R. Beckwith, A Deletion ~naylsis of the ~ac Operator Region in Escherichia coli, ~, Mol. BiDl,~ B ~1964) pp 427-430.
Tai-kin Wong, Attempts to Characterize the aLate~Template"
Properties of T5 DNA after Infection of E, ~bli and Some Aspects on the Mechanism of Plasmid pSC101 M~ ated Te~ra-eycline R~sistance, Ph.D. Dissertation, Ruprecht-Xarl-Universitat Heidelberg, lg78. West Germany.
9~.N. Timmis, F, Cabello, S,N, Cohen, Cloning and Characterization of EcoRI and HinaIII Restriction Endonuclease-generated Fragments of Antibiotic Resistanc~ Plasmi~s R6 5 and R6, ~olec. Gen.
Genet. 162 ~1978) pp. 121-137.
10E, Colbere-Garapin, S. Chousterman, ~, Horodniceanu, ~.
Kourilsky, ~.C. Garapin, Cloning of the Ac~ive Thymidine Xinase Gene of Herpes Sl~mplex Virus ~ype 1 in Escherichia coli, Proc Natl. Acad. Sci. ~SA, 76 tlg79) pp. 3755~3759.
llGibco~ Grand I~land Biological Company, Catalog No, 430-1800.
~9 i~2~
oxoid Limited, En~land~ Code BR14a, 13RPMI 1640 Supplemented with Hypoxanthine 13.6 ~g/ml; .
Aminopterin 0.176 ~g/ml; thymidine 3,78 yg/mlt modified from E.B. 5zybalska, W. Szybalski, Genetics of ~uman Cell Lines. IV. DNA-mediated Heritable Transform~tion of a Bioch~mical Trait, Proc. Natl. Acad. Sci. USA, 48: (1962) pp. 2026-2034, MacConkey Agar Supplemented with 20mM IP~G, ~Isopropyl-~-D-thiogalactopyranoside~ Difco, Catalog No~ 00~5-05, IPTG: Sigma 1-5502.
Claims (14)
1. The method of transferring genes into cells which comprises the step of subjecting a mixture of said genes and said cells to an electric field.
2. The method according to Claim 1 wherein the said electric field comprises an electric discharge.
3. A method according to Claim 1 wherein said mixture is a suspension containing said cells and said genes.
4. A method according to Claim 1 wherein said electric field is from 3 kilovolts to about 20 kilovolts.
5. A method according to Claim 3 wherein said cells are procaryotic cells.
6. A method according to Claim 3 wherein said cells are eucaryotic cells.
7. A method according to Claim 3 wherein said cells are Streptomyces.
8. A method according to Claim 3 wherein said cells are plant cells.
91 A method according to Claim 3 wherein said cells are mammalian cells.
10. A method according to Claim 3 wherein said genes comprise a piece of DNA.
11. A method according to Claim 3 wherein said genes are transferred into said cells on plasmid vectors.
12. A method according to Claim 3 wherein said genes are transferred into said cells on bacteriophage vectors,
13. A method according to Claim 3 wherein said genes are transferred into said cells on viral vectors.
14. A method according to Claim 3 wherein said genes are transferred into said cells on yeast vectors.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000439852A CA1208146A (en) | 1983-10-27 | 1983-10-27 | Method of transferring genes into cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000439852A CA1208146A (en) | 1983-10-27 | 1983-10-27 | Method of transferring genes into cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1208146A true CA1208146A (en) | 1986-07-22 |
Family
ID=4126389
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000439852A Expired CA1208146A (en) | 1983-10-27 | 1983-10-27 | Method of transferring genes into cells |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1208146A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0290395A2 (en) * | 1987-05-05 | 1988-11-09 | Sandoz Ag | Plant tissue transformation |
| EP0319690A1 (en) * | 1987-11-24 | 1989-06-14 | Nisshin Flour Milling Co., Ltd. | Method of introducing foreign DNA into certain bacteria |
| US5231019A (en) * | 1984-05-11 | 1993-07-27 | Ciba-Geigy Corporation | Transformation of hereditary material of plants |
| US5232856A (en) * | 1990-06-25 | 1993-08-03 | Firth Kevin L | Electroporation device |
| EP0624100A1 (en) * | 1992-11-03 | 1994-11-17 | Synaptic Pharmaceutical Corporation | Dna encoding a human serotonin receptor (5-ht 4b?) and uses thereof |
| US5371003A (en) * | 1987-05-05 | 1994-12-06 | Sandoz Ltd. | Electrotransformation process |
| US6300087B1 (en) | 1992-11-03 | 2001-10-09 | Synaptic Pharmaceutical Corporation | DNA encoding a human serotonin receptor (5-HT4B) and uses thereof |
-
1983
- 1983-10-27 CA CA000439852A patent/CA1208146A/en not_active Expired
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6201169B1 (en) | 1984-05-11 | 2001-03-13 | Novartis Finance Corporation | Transformation of hereditary material of Brassica plants and cells |
| US5231019A (en) * | 1984-05-11 | 1993-07-27 | Ciba-Geigy Corporation | Transformation of hereditary material of plants |
| US6603065B2 (en) | 1984-05-11 | 2003-08-05 | Syngenta Invest Corp | Transformation of hereditary material of plants |
| EP0290395A2 (en) * | 1987-05-05 | 1988-11-09 | Sandoz Ag | Plant tissue transformation |
| EP0290395A3 (en) * | 1987-05-05 | 1990-11-28 | Sandoz Ag | Plant tissue transformation |
| EP0574356A1 (en) * | 1987-05-05 | 1993-12-15 | Sandoz Ag | Plant tissue transformation |
| US5371003A (en) * | 1987-05-05 | 1994-12-06 | Sandoz Ltd. | Electrotransformation process |
| EP0319690A1 (en) * | 1987-11-24 | 1989-06-14 | Nisshin Flour Milling Co., Ltd. | Method of introducing foreign DNA into certain bacteria |
| US5232856A (en) * | 1990-06-25 | 1993-08-03 | Firth Kevin L | Electroporation device |
| EP0624100A1 (en) * | 1992-11-03 | 1994-11-17 | Synaptic Pharmaceutical Corporation | Dna encoding a human serotonin receptor (5-ht 4b?) and uses thereof |
| EP0624100A4 (en) * | 1992-11-03 | 1995-11-29 | Synaptic Pharma Corp | Dna encoding a human serotonin receptor (5-ht 4b?) and uses thereof. |
| US6376243B1 (en) | 1992-11-03 | 2002-04-23 | Synaptic Pharmaceutical Corporation | DNA encoding a human serotonin receptor (5-HT4b) and uses thereof |
| US6432655B1 (en) | 1992-11-03 | 2002-08-13 | Synaptic Pharmaceutical Corporation | Method of obtaining compositions |
| US6300087B1 (en) | 1992-11-03 | 2001-10-09 | Synaptic Pharmaceutical Corporation | DNA encoding a human serotonin receptor (5-HT4B) and uses thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4849355A (en) | Method of transferring genes into cells | |
| Chang et al. | Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid | |
| Fishel et al. | recA-independent general genetic recombination of plasmids | |
| JP2588917B2 (en) | Microorganisms and plasmids for the formation of 2,4-dichlorophenoxyacetic acid (2,4-D) -monooxygenase and methods for preparing these plasmids and strains | |
| Hershfield et al. | Characterization of a mini-ColC1 plasmid | |
| Fleckenstein et al. | Cloning of the complete human cytomegalovirus genome in cosmids | |
| Meyer et al. | Physical and genetic studies with restriction endonucleases on the broad host-range plasmid RK2 | |
| Ball et al. | Efficient excision of phage lambda from the Escherichia coli chromosome requires the Fis protein | |
| Rella et al. | Transposon insertion mutagenesis of Pseudomonas aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene cluster | |
| US5869296A (en) | DNA transposon Tn5seq1 | |
| Huber et al. | Site-specific DNA inversion is enhanced by a DNA sequence element in cis | |
| Bonekamp et al. | Mechanism of UTP‐modulated attenuation at the pyrE gene of Escherichia coli: an example of operon polarity control through the coupling of translation to transcription. | |
| HU197043B (en) | Process for the stabilization and selection of host-cells comprising recombinant dna | |
| Sakakibara et al. | A temperature-sensitive Escherichia coli mutant defective in DNA replication: dnaA, a new gene adjacent to the dnA, gene | |
| Prasad et al. | Mutations in the 2-microns circle site-specific recombinase that abolish recombination without affecting substrate recognition. | |
| Kahn et al. | Essential regions for replication of a stringent and a relaxed plasmid in Escherichia coli | |
| CA1208146A (en) | Method of transferring genes into cells | |
| Howe | Transduction by bacteriophage Mu-1 | |
| Gryczan et al. | Direct selection of recombinant plasmids in Bacillus subtilis | |
| Shattuck-Eidens et al. | Molecular cloning of the uhp region and evidence for a positive activator for expression of the hexose phosphate transport system of Escherichia coli | |
| Kooistra et al. | Cloning and characterization of a Bacillus subtilis transcription unit involved in ATP-dependent DNase synthesis | |
| Bonamy et al. | Cloning and expression of Bacillus subtilis spore genes | |
| Löfdahl et al. | Characterization of small plasmids from Staphylococcus aureus | |
| US4722897A (en) | Viral enhancer DNA segments | |
| Lacks et al. | Cloning in Streptococcus pneumoniae of the gene for DpnII DNA methylase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |