Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-05-11
2001-12-18
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S320100, C435S091400, C435S091500, C536S023100
Reexamination Certificate
active
06331397
ABSTRACT:
The present invention relates to a method for producing long DNA constructs, particularly artificial chromosomes, and vectors usable for this purposes, as well as a method of providing large DNAs, particularly BACs or PACs.
Modern research considers to produce therapeutic proteins in animal cells and transgenic animals, respectively. It is also considered to repair defects of animal, particularly human, cells. In particular, there are considerations of compensating defective genes by corresponding healthy ones. To this end, it is proposed e.g. to integrate the healthy genes in an expressible form into the genome of the cells. It is also taken into account to apply the healthy genes on mammalian (human) artificial chromosomes (hereinafter referred to as MACs (HACs)) and introduce them into the cells. MACs are linear DNAs having a size of several 100 kb. They distinguish themselves by various components. These are per MAC one centromer, two terminal telomeres and a chromosome arm therebetween having at least one origin of replication. The above genes can be applied to the latter component.
However, the production of MACs is accompanied by major problems. Experiments carried out so far to achieve such a production have not been satisfactory In particular, the size of the individual components represents a problem for cloning.
Therefore, it is the object of the present invention to provide a method by which it is possible to produce long DNA constructs, particularly MACs.
According to the invention this is achieved by the subject matters defined in the claims. Thus, the subject matter of the present invention relates to a method and vectors by which long DNA constructs, particularly MACs, can be prepared. A further subject matter of the present invention relates to a method by which large DNAs, e.g. BACs “bacterial artificial chromosomes” or PACs “phage P
1
artificial chromosomes”, particularly components of MACS, can be provided in great amount and stability.
The present invention is based on the applicant's insights that large DNAs, such as BACs or PACs, can be recombined with one another in melted agarose. He found that MACs can be produced by this if the large DNAs have the individual components of MACs, such as a centromer, e.g. an alpha-satellite DNA, two telomeres, e.g. the sequence (TTAGGG)
n=135
, and a chromosome arm containing at least one ‘origin of replication’. He also recognized that bacteria which contain large DNAs, such as BACs or PACs, can be mixed with agarose, so that little agarose blocks are obtained when the agarose has cooled down. The chromosomal DNA of the bacteria can be cleaved therein, while the large DNAs remain uncleaved. To this end, a restriction enzyme is inserted in the little blocks, which cleaves exclusively the bacterial chromosome. It can then be removed from the little blocks by gel electrophoresis, so that only the large DNAs remain in the little blocks. The applicant recognized that the large DNAs as well as the DNA constructs are stable in the little agarose gel blocks and can be stored over a long period of time.
According to the invention, the applicant's insights are used for a method of preparing DNA constructs, comprising the combination of two DNAs by means of recombination in melted agarose.
The expression “DNA construct” refers to a DNA of any kind and length, which can be circular or linear. For example, the DNA is linear and has a length of several 100 kb. The DNA is preferably an artificial mammalian chromosome (MAC).
It is particularly preferred for the MAC to comprise one or several genes whose expression is desired. Examples of such genes are those which in a defective form are connected with diseases, e.g. mucoviscidosis.
The expression “combination by means of recombination” refers to the fact that two DNAs can be recombined with each other. This can be effected by overlapping sequences. It may be favorable for the sequences to comprise recombination-specific sequences such as lox or FRT sequences, the recombination then occurring in the presence of a recombinase, such as Cre or Flp recombinase. The recombination of the two DNAs preferably results in a MAC. In this case, the DNAs comprise all of the elements important for a MAC. Furthermore, they can have one or several genes whose expression is desired. In this connection, reference is made to the above explanations. For example, if a MAC is combined from two DNAs, it can be favorable for one of the DNAs to be present in linear form and the other to be present in circular form. The latter may contain one or several genes whose expression is desired. The linear DNA can contain a centromer and terminal telomeres, which have opposite orientation. The two DNAs can also be present in linear form, each DNA having a telomere which in one DNA is present at the left end and in the other DNA is present at the right end in opposite orientation each. Moreover, the two DNAs can be present in circular form, the circular recombination product having to be linearized by means of recombination cleavage for the development of a MAC.
A further subject matter of the present invention relates to vectors which contain one or several, particularly two, telomeres. Such vectors are suitable to carry out the method according to the invention. Preferred vectors are those which have two telomeres with opposite orientation, e.g. the sequence (TTAGGG)
n=135
, and a recombination-specific sequence located between the telomere beginnings, e.g. lox sequence. Special preference is also given to vectors which also have two resistance genes, one, e.g. kanamycin, being located between the telomere ends, and the other, e.g. ampicillin, being located between the telomere beginnings. Vectors which also have recognition sequences for restriction enzymes which are rare and/or cleave once and several times, respectively, between the telomere beginnings and/or ends are preferred as well. Particularly preferred vectors are the ditelomeric vector PTAT and the monotelomeric vectors PT1, PT1L, PT1LA and PT1LAS. In this connection, reference is made to the examples. The vectors according to the invention can be present as such or in a kit. It can also contain a recombinase, such as Cre or FLP recombinase, and common auxiliary substances, such as buffers, solvents, etc.
A further subject matter of the present invention relates to a method of providing large DNA, particularly BACS or PACs. Such a method comprises the steps of:
(a) mixing a bacterial culture with melted agarose so as to obtain little agarose blocks when the agarose has cooled down,
(b) introducing one or several restriction enzymes into the little agarose blocks, the restriction enzymes only cleaving the bacterial chromosome but not the large DNA, and
(c) carrying out gel electrophoresis so as to remove the cleaved bacterial chromosome from the little agarose blocks, while the large DNA is retained.
The expression “melted agarose” refers to what is called a ‘low melting agarose’ which melts at a low temperature.
The expression “gel electrophoresis” refers to the fact that the little agarose blocks are subjected to common gel electrophoresis, particularly pulsed field gel electrophoresis. In this connection, reference is made to the examples. The expression “large DNA” comprises large extra-chromosomal DNA, e.g. BACs or PACs.
By means of the present invention it is possible to produce long DNA constructs, particularly artificial chromosomes. They can contain desired genes. Furthermore, they can be stored in stable fashion for a long time. Moreover, they can be varied as desired and be rapidly adapted to new requirements. Reference is made explicitly to the examples. Therefore, the present invention represents a breakthrough for the well-calculated treatment of defective genes.
REFERENCES:
The 1998/1999 BioRad Catalog, pp 219-220, 1999.*
Larin, et al. Methods of Molecular Biology, vol. 54, pp. 1-11, 1995.
Cooke Howard
Schindelhauer Dirk
Guzo David
Hultquist Steven J.
Leffers, Jr. Gerald G.
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