Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2002-01-15
2003-07-01
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S470000, C435S471000, C435S488000, C435S252300, C435S256300, C435S440000, C435S410000, C435S325000, C435S254210
Reexamination Certificate
active
06586249
ABSTRACT:
The invention relates to the field of electroporation and transformation of host cells, particularly bacterial cells.
BACKGROUND OF THE INVENTION
Introducing nucleic acids into
E. coli
hand other host organisms is central to many types of experiments and analyses. For example, when searching for a gene of interest in a DNA library, the library must be transferred into a host organism. Since the DNA of many organisms is very complex, the number of independent clones that are needed to completely represent the organism is large. In order to create a library that completely represents the organism, the efficiency at which the DNA can be introduced into the host cell becomes limiting. By optimizing this process, the ability to create and screen DNA libraries is facilitated.
Similarly, many other experimental analyses are limited by the ability to introduce DNA into a host organism. When cloning large segments of DNA for whole genome analysis (i.e., using bacterial artificial chromosomes), when performing PCR cloning, or when carrying out random mutagenesis of a gene, followed by cloning all potential altered forms, success often depends on the size of the initial transformation pool. Again, developing conditions that improve the process of introducing nucleic acids into a host organism increases the chance that the experiment will succeed.
There are several methods for introducing nucleic acids into various host cells, e.g., incubating the host cells with co-precipitates of nucleic acids (Graham and van der Eb,
Virology,
52: 456-467 (1973)), directly injecting genes into the nucleus of the host cells (Diacumakos,
Methods in Cell Biology,
Vol. 7, eds. Prescott, D. M. (Academic Press) pp. 287-311 (1973), introducing nucleic acids via viral vectors (Hamer and Leder,
Cell,
18: 1299-1302 (1979)), and using liposomes as a means of gene transfer (Fraley et al.,
J. Biol. Chem.,
255: 10431-10435 (1980); Wong et al.
Gene,
10: 87-94 (1980)). Electroporation has also been used to transform host organisms, including
E. coli.
(Dower et al.,
Nucleic Acids Research,
16: 6127-6145 (1988); Taketo,
Biochimica et Biophysica Acta,
949: 318-324 (1988); Chassy and Flickinger,
FEMS Microbiology Letters,
44: 173-177 (1987); and Harlander,
Streptococcal Genetics,
eds. Ferretti and Curtiss (American Society of Microbiology, Washington, D.C.) pp. 229-233 (1987)).
In general, electroporation involves the transfer of genes or gene fragments (nucleic acids) into a host cell by exposure of the cell to a high voltage electric impulse in the presence of the genes or gene fragments (Andreason and Evans,
Biotechniques,
6: 650-660 (1988)). Quite often, the genes and gene fragments are exogenous, i.e., heterologous to the host organism. Also, frequently the cells have been stored prior to electroporation. A typical method of storage is to freeze the cells. The cells are frozen at a temperature that preserves viability. After thawing those cells, genes or gene fragments may be transferred by electroporation into the cells, permanently or transiently for short-term expression.
An example of a typical electroporation method is to grow bacteria in enriched media (of any sort) and to concentrate the bacteria by washing in a buffer that contains 10% glycerol (Dower et al., 1988, U.S. Pat. No. 5,186,800). As discussed in U.S. Pat. No. 5,186,800, which is hereby incorporated by reference in its entirety, DNA is added to the cells and the cells are subjected to an electrical discharge, which temporarily disrupts the outer cell wall of the bacterial cells to allow DNA to enter the cells.
The electrical treatment to which the host cells are subjected during the process of electroporation is very harsh and typically results in the death of >90% of the host cells. However, it is believed that the majority of cells that survive electroporation take up the nucleic acids of interest. The efficiency with which nucleic acid transfer occurs depends on a variety of factors, including the genetic background of the host cells. Routinely, an efficiency of 10
9
−1×10
10
transformants per &mgr;g of input DNA (plasmid pUC18) may be achieved. Using Rec A− cells, typically 5.0-7.0×10
9
cells are transformed per &mgr;g of input DNA. When the host cells are
E. coli,
10% or less of the treated bacteria survive. However, the percentage is significantly lower for certain strains of
E. coli
that are inefficient at electrotransformation.
In developing and refining electroporation methodology, researchers have identified factors that impact the efficiency of the transfer. These factors include, e.g., the electrical field strength, the pulse decay time, the pulse shape, the temperature in which the electroporation is conducted, the type of cell, the type of suspension buffer, and the concentration and size of the nucleic acid to be transferred (Andreason and Evans,
Analytical Biochemistry,
180: 269-275 (1988); Sambrook, et al.,
Molecular Cloning: a Laboratory Manual,
2nd Edition, eds. Sambrook, et al. (Cold Spring Harbor Laboratory Press) pp. 1.75 and 16.54-16.55 (1989); Dower et al., (1988); Taketo (1988)). Thus, previous attempts to improve the electroporation efficiency have focused on these factors and thus, have primarily involved manipulation of methods used to prepare the cells, e.g., washing and centrifugation of cells during the processing stage, and methods for applying the electrical shock (i.e., different configuration of the apparatus that delivers the electrical pulse).
Typically, researchers have only modified the host cell suspension materials to aid in freezing the cells before the electrical treatment (Taketo 1988).
SUMMARY OF THE INVENTION
This invention provides improved methods of electroporation and other electrical treatment of cells. The methods comprise the addition of sugars or sugar derivatives, e.g., sugar alcohols, to host cells suspended in a substantially non-ionic solution, either prior to initial freezing, or after thawing, but prior to electrotransformation. The methods of this invention improve electroporation efficiency. The level of improvement is 30% (for cells that generally exhibit higher efficiency) to 300% (for cells that have lower efficiency).
In certain embodiments, at least one sugar or sugar derivative is added to the host cells suspended in a substantially non-ionic solution prior to electrically treating the host cells. Preferably, the sugar or derivative thereof is added in a concentration range of about 0.1% to about 5%. In certain embodiments of the invention, the host cells are suspended in the non-ionic sugar or sugar derivative solution before they are electrically transformed. In certain embodiments, one may prefer to freeze the cells prior to the electrotransformation For instance, one may suspend the host cells in the sugar or sugar derivative solution before freezing the cells. In another instance, the cells may be suspended in the sugar or sugar derivative solution after the cells have been frozen and thawed. In certain embodiments, the host cells are bacterial cells, preferably gram-negative bacterial cells, and most preferably,
E. coli.
This invention also provides a kit for use in the practice of the above-described methods of transferring nucleic acids of interest into host cells. The electroporation kit includes host cells suspended in a substantially non-ionic solution comprising at least one sugar or sugar derivative. In certain embodiments, the kit includes host cells suspended in a non-ionic solution having a sugar or sugar derivative a concentration of about 0.1% to about 5%. In other embodiments, the kit includes a non-ionic solution comprising a mixture of sugars and sugar derivatives.
DETAILED DESCRIPTION OF THE INVENTION
Thus, this invention provides methods for transferring nucleic acids of interest into host cells, comprising the steps of mixing host cells suspended in a substantially non-ionic solution comprising at least one sugar or sugar derivative with nucleic acids of interest, and subjecting the mixture to ele
Greener Alan L.
Jerpseth Bruce D.
Finnegan Henderson Farabow Garrett & Dunner LLP
Ketter James
Stratagene
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