Selection for more efficient transformation host cells

Details Selection for more efficient transformation host cells Selection for more efficient transformation host cells

2000-07-26

2003-10-21

Yucel, Remy (Department: 1636)

Chemistry: molecular biology and microbiology

Treatment of micro-organisms or enzymes with electrical or...

Metabolism of micro-organism enhanced

C435S173100, C435S440000, C435S471000, C435S455000, C435S468000, C435S252100, C435S325000, C435S410000

Reexamination Certificate

active

06635457

ABSTRACT:

BACKGROUND AND SUMMARY OF THE INVENTION
The invention relates to the field of transformation of host organisms.
Introducing nucleic acids into
E. coli
and 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 make a library that completely represents the organism, the efficiency at which the DNA can enter the host cell becomes limiting. Optimizing this process facilitates the ability to create and screen DNA libraries.
a Similarly, for many other experimental approaches, the ability to introduce DNA into a host organism is the limiting factor. When cloning large segments of DNA for whole genome analysis (i.e., using bacterial artificial chromosomes), when performing PCR cloning (sometimes an inefficient process), or when carrying out random mutagenesis of a gene followed by cloning all potential altered forms, success often depends on the primary size of the initial transformation/electroporation pool. Again, by developing conditions that improve the process of introducing nucleic acids into any host organism, one increases the chance that the experiment will succeed.
There are several methods for introducing nucleic acids into host cells, e.g., incubating the host cells with co-precipitates of nucleic acids and Ca-phosphate (an example of chemical transformation) (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, 1973) pp. 287-311), 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 is another method for introducing nucleic acids into eukaryotic cells (Neumann et al.
Embo J.
1: 841-845 (1982)). The method has also been used in transforming
E. coli
(Dower et al.,
Nucleic Acids Research
16: 6127-6145 (1988); Taketo,
Biochimica et Biophysica Acta
949: 318-324 (1988)) and other bacteria (Chassy and Flickinger,
FEMS Microbiology Letters
44: 173-177 (1987); Harlander,
Streptococcal Genetics
, eds. Ferretti and Curtiss (American Society of Microbiology, Washington, D.C. 1987) pp. 229-233).
Indeed, electroporation has become a typical method for transferring nucleic acids into a host organism. This method of transformation is generally performed by subjecting a host to a very strong electrical discharge, which typically kills a majority of cells. Only a small percentage survive. The process of electroporation is harsh and often results in the death of about 90% or more of the host organisms.
Electroporation involves the transfer of genes or fragments of nucleic acids into a host cell by exposure of the cell to a high voltage electric impulse in the presence of the genes or fragments (Andreason and Evans,
Biotechniques
6: 650-660 (1988)). Quite often, the genes and fragments of nucleic acids are exogenous. If the cells survive the electroporation, it has been confirmed that over 90% of those cells will take up the nucleic acids. With this level of transfer, electroporation has been used to introduce genes or fragments of nucleic acids into host cells both permanently or transiently for short-term expression.
One typical electroporation protocol involves growing bacteria in rich media and concentrating them 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 then added to the cells, and the cells are subjected to an electrical discharge, which disrupts the outer cell wall of the bacterium and allows DNA to enter the cell. The efficiency of transfer in
E. coli
varies depending on factors, including the genetic background of the
E. coli
. Routinely, an efficiency of 1×10
9
to 1×10
10
transformants per &mgr;g of input DNA may be achieved (XL-1Blue™ and XL10-Gold™, both cell lines from Stratagene).
A limitation on previous electroporation methods involving prokaryotic cells is that generally RecA-deficient cells are used. While RecA-deficient cells are stable host cells for transformation, cells expressing RecA show a high incidence of homologous recombination. Therefore, using RecA-expressing cells typically can cause problems with electroporation because the introduced nucleic acids would produce highly unstable transformants.
However, due to the deficiency, RecA-deficient cells do not survive the electroporation process as well as RecA-expressing cells. Therefore, those skilled in the art must choose between using RecA-deficient cells, which would be ideal for transformation experiments but often die before being transformed, and RecA-expressing cells, which survive transformation but typically produce unstable transformants. Despite the low survival rates, those of skill in the art typically use RecA-deficient cells.
Previous attempts to improve the electroporation process have involved methods used to prepare the cells, e.g., washing and centrifuging of cells during the processing stage and methods for applying the electrical shock, (e.g., different configuration of the apparatus that delivers the electrical pulse). The specific conditions that have been adjusted include, e.g., the concentration of nucleic acids, the temperature, the pulse decay time, and the initial field strength. Another attempt involves using different suspension materials to stabilize the cells in solution and assist in freezing them before the electrical treatment (Taketo 1988).
Another known method of introducing nucleic acids into a host organisms is by chemical treatment of the host organism. Typically, chemicals have been used to transfer nucleic acids into host cells, e.g., transfection. Known methods of chemically-mediated nucleic acid transfer are calcium phosphate-mediated transfection, and a variation, DEAE-dextran-mediated transfection (Sambrook et al.
Molecular Cloning: a laboratory manual,
2
nd
edition
, eds. Sambrook et al. (Cold Spring Harbor Laboratory Press 1989)). Other chemically-mediated transformation methods include: 1) using polybrene to introduce the nucleic acids into cells that are resistant to other methods of transfection (Kawai and Nishizawa,
Mol. Cell Biol.
4: 1172-1174 (1984); Chaney et al.,
Somat. Cell Mol. Genet.
12: 237-244 (1986)); 2) using polyethylene glycol to fuse protoplasts with cultured mammalian cells (Schaffner et al.,
PNAS, USA
77: 2163-2167 (1980); Rassoulzadegan et al.,
Nature
295: 257-259 (1982)); and 3) coating the nucleic acids of interest with a synthetic cationic lipid to introduce them into cells by fusion (Felgner et al.,
PNAS, USA
84: 7413-7417 (1987)).
According to certain embodiments, the present invention is directed to cells and methods of making cells that are more efficiently transformed in electroporation processes. The methods of the invention provide cells that are better able to survive and to be transformed than cells presently used in electroporation transformation methods.
As noted above, the process of electroporation according to known processes generally kills more than 90% of the host organisms (with the majority of the surviving cells taking up the DNA). According to certain embodiments of the invention, methods are provided in which host cells are altered and screened by exposing the altered cells to typical electroporation conditions. One selects altered cells that are able to survive the transformation conditions, e.g., electrical discharges. The selected altered cells have better viability after exposure to electroporation and are better suited to take up nucleic acids by electroporation. Thus, the alter

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