Note: Descriptions are shown in the official language in which they were submitted.
`. " ~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
-3-
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.
4--
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
TABLE 1
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~
-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~
12
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.
