Selection marker

Details Selection marker Selection marker

1998-06-04

2001-10-30

Patterson, Jr., Charles L. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S320100, C435S195000, C536S023200

Reexamination Certificate

active

06309874

ABSTRACT:

The present invention relates to a new method for selecting cells which involves a mutated naturally occuring life-essential enzyme, namely a (Na,K)-ATPase (EC 3.6.1.3). A mutated naturally occuring (Na,K)-ATPase or a subunit thereof, as well as their medical use is claimed. Also transgenic animals are claimed.
When transfecting genes to cells or individuals, a selection is often required and multiplication of the cells which have received the new gene is desirable. This can be done in many ways. Today a usual procedure is to simultaneously use genes which are resistant to antibiotics or cytostatics which makes the cells with the new gene resistant to these substances. It is then possible to make a selection between host cells which have received the intibiotic resistance gene and those host cells who have not by using the corresponding antibiotic. This is a well-known procedure for a person skilled in the art. (See e.g. Gene transfer and expression protocols. (Murray E. J, ed) Humana Press, New Jersey)
This procedure however have some disadvantages. It is expensive and may disturb the host cell due to the fact that an artificial gene has to be put in together with a certain selected gene. The cells must be selected using antibiotics or cytostatics. These antibiotic and cytostatic compounds might also interfere with other cellular mechanisms. It is not suitable to insert genes confering antibiotic or cytostatic resistance to human cells in the case of gene therapy. A current problem with gene therapy today is that the efficiency is low; possibly due to the low level of gene transfected cells.
Accordingly, there is a need for a new method for selection of cells which lack these disadvantages.
SUMMARY OF THE INVENTION
It has now been found, that by using a protein comprising the amino acid sequence: IFIIANIPXPXGTVTIXXID (SEQ ID NO:9) where X is chosen from the group of amino acids: cysteine, leucine, glycine, alanine, valine, isoleucine, where at least one X is cysteine, as a marker for screening cells, most of the drawbacks listed above can be overcome. Preferably the protein comprises at least one of the following three amino acid sequences: IFIIANIPCPLGTVTILCID (SEQ ID NO:10), IFIIANIPLPCGTVTILCID (SEQ ID NO:11) or IFIIANIPCPCGTVTILCID (SEQ ID NO:12).
Accordingly a new selection system fulfilling the above mentioned needs has now been discovered. The system is based upon using a protein already existing in the cell protein as selection gene namely (Na,K)-ATPase, EC 3.6.1,3. This enzyme is essential for the cell and the gene is expressed in all mammalian cells. The (Na,K)-ATPase (a.k.a. Na+,K+-ATPase or NKA) is essential for the cell and it transports the sodium and potassium ions over the cell membrane. Without the function of this enzyme the cell will die
In this system, such a gene has been mutated in a particular manner which causes a loss in sensitivity to the substance ouabain (a cardiotonic steroid or glycoside). This substance is able to kill all cells which have not received the mutated protein and surviving cells are only cells which have received the mutated gene product. Ouabain normally depresses the activity of (Na,K)-ATPase.
There are advantages with this new system. No heterologous gene has to be added to the cells. This minor mutation is more natural than adding an artificial gene or making some major mutations. Moreover, the selection substance ouabain is also relatively cheap and stable. Ouabain is neither an antibiotic substance nor a cytostatic compound. In gene therapy, it will not be necessery to screen using antibiotic or cytostatic compounds. The possibilty is also given to purify and multiply only interesting cells for gene therapy (e.g. heinatopoetic stemcells) with minimal intervention in the natural system of the cell. This new system gives an improved possibility of transporting new genes into cells from humans and animals. This can also improve the results in gene therapy and transplantation of e.g. hematopoetic stem cells, which have been subjected to gene therapy, to humans. Due to the fact that selection can be carried out with an enzyme, preferably NKA, which is already situated in the cell, an immunological answer on the transfected cells is minimized or eliminated when the cells are transplanted into a patient, e.g. when stem cell transplanting stem cells to a to human. The risk for an immunological reaction to occur must be regarded to be much higher when using a selection gene which is normally not expressed in the cell. There is also a new possibility to study effects of cardiac glycosides (e.g. digitalis) on cells or whole animals who are resistant to these drugs. This ouabain-resistant enzyme could also be very useful when studying the enzyme or its biological effects.
We will now go into further details of the invention.
DETAILED DESCRIPTION OF THE INVENTION
The protein encoded by the gene as well as variants, subfragments and multiples of the protein having essentially the same antigenic and/or binding characteristics also constitutes an object of the present invention. The new protein is referred to as 799/801NKA. Preferably the amino acid sequence of the 799/801NKA should be at least 70% homologous, more prefarbly at least 85% homologous, still more preferably at least 90% homologous and most preferably at least 95% homologous to anyone of the amino acid sequences disclosed in SEQ ID NOS 1, 2, 3. Preferably 799/801NKA has either a cysteine in position 799 or in position 801 or a cysteine in both positions. When we call the protein life-essential later in this document we mean that the cell can not survive without its function.
By “subfragments” is meant a part-fragment of the given protein in having essentially the same antigenic and /or binding characteristics. By “variants” is meant proteins or peptides in which the original amino acid sequence has been modified or changed by insertion, addition, substitution, inversion, or exclusion of one or more amino acids. By “multiples” is meant those proteins containing multiples of the whole original protein or those protein containing multiples of subfragments and/or variants thereof.
The present invention also relates to nucleic acid sequences encoding 799/801NKA As utilized within the context of the present invention, nucleic acid sequences which encode 799/801NKA are deemed to be substantially similar to those disclosed herein if; (a) the nucleic acid sequence is derived from the coding region of a native NKA gene (including, for example, variations of the sequences disclosed herein); (b) the nucleic acid sequence is capable of hybridization to nucleic acid sequences of the present invention under conditions of either moderate or high stringency (hybridization in 5×SSPE containing 0 1% SDS and 0.1 mg/ml ssDNA, at 50-65°/C. dependent on the probe length, or 10-20°/C. below the T
m
of the probe; washing in 1×SSPE, 0.1% SDS at 1 5-20°/C. below the T
m
of the probe for moderate stringency, and in 0.1×SSPE, 0.1% at 10°/C. below the T
m
of the probe for high stringency conditions) (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, NY, 1989); or (c) nucleic acid sequences are degenerate as a result of the genetic code to the nucleic acid sequences defined in (a) or (b). Furthermore, although nucleic acid molecules are primarily referred to herein, as should be evident to one of skill in the art given the disclosure provided herein, a wide variety of related nucleic acid molecules may also be utilized in various embodiments described herein, including for example, RNA, nucleic acid analogues, as well as chimeric nucleic acid molecules which may be composed of more than one type of nucleic acid.
Within another aspect of the present invention, probes and primers are provided for detecting nucleic acids sequences which encode 799/801NKA. Within one embodiment of the invention, probes are provided which are capable of hybridizing to 799/801NKA nucleic acids (DNA or RNA). For purposes of the present invention,

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