DE10016523A1 - Cloning system for the construction of homologous recombination vectors - Google Patents

Abstract

The invention relates to a novel cloning system that consists of a vector and adapter system that substantially simplifies the construction of homologous recombination vectors used for the mutagenesis of genes in living eukaryotic cells. The invention further relates to a method for producing homologous recombination vectors as well as to the use of said cloning system for modifying the genome at defined loci in eukaryotic cells, especially in embryonic stem cells.

Description

The invention relates to a new cloning system consisting of a vector and Adapter system for the construction of homologous recombination vectors Mutagenesis of genes in living eukaryotic cells considerably simplified. Farther The invention relates to a method for producing homologous recombination vectors. In addition, the invention relates to the use of the cloning system to in eukaryotic cells, especially embryonic stem cells, defined the genome Change loci.

Numerous methods are described in the literature, such as using chromosomal methods Changes in genes whose expression or structure can be changed. (Capechi, M.R. (1989), Altering the genome by homologous recombination, Science 244: 1288-1292; Bradley, A., Hasty, P., Davis, A. and Ramirez-Solis, R. (1992), Modifying the mouse: design and desire, Bio / Technology 10: 534-539.)

Targeted mutagenesis is achieved using the homologous recombination method carried out. A synthetic vector, which is introduced into a cell, recombines Contains parts of the chromosomal locus to be modified, with the genomic DNA of the Cell. (Clarke, A.R. (1994), Murine genetic models for human diseases, Curr. Opin. Genet. Dev. 4: 453-460; Melton, D.W. (1994), Gene targeting in the mouse, Bioassays 16: 633-638.)

The isolation of the cells into which the vector DNA has been integrated chromosomally from the pool non-recombined cells are made possible by a positive selection cassette in the vector, which is flanked by the chromosomal vector DNA. Any stable integration of the vector DNA leads to permanent resistance to a cytotoxic pharmacological agent, which is expressed by a specific resistance gene. Examples are the  Resistance to geniticin (G418) through the integrated neomycin gene or resistance against hygromycin through the integration of hygromycin gene.

The inserted positive selection cassette can be selected by the choice of the insertion in the chromosomal vector DNA to a mutation of the gene in the sense of a classic knock outs, that is, inactivation of gene function. Deactivation or modification of a regulating genetic element influencing gene expression or functional part of the transcribed / translated gene product can both have positive, negative, as well as modifying influence on the gene function. (Bradley, A., Hasty, P., Davis, A. and Ramirez-Solis, R. (1992), Modifying the mouse: design and desire, Bio / Technology 10: 534-539; Van der Neut, R. (1997), Targeted gene disruption: Applications in Neurobiology J. Neurosci. Methods 71: 19-27.)

The desired homologous recombination, which runs over the positive selection cassette, must counteract the unwanted non-homologous recombination that ends over the vector runs, be selected. To do this, the vector ends with negative Selection cassettes equipped, which then often in the non-homologous recombination ins Genome to be integrated (Mansour, S.L., Thomas, K.R. and Capechi, M.R. (1988), Disruption of the proto-oncogene int-2 in mouse embryo derived stem cells: a general strategy for targeting mutations to non-selectable genes, Nature 336: 348-352). One usually non-cytotoxic agent becomes stable expression of the negative selection cassettes cytotoxic. An example of this is the activated cytotoxicity of gancyclovir by the herpes simplex virus thymidine kinase gene (HSV-tk) (Mansour, S.L., Thomas, K.R. and Capechi, M.R. (1988), Disruption of the proto-oncogene int-2 in mouse embryo derived stem cells: a general strategy for targeting mutations to non- selectable genes, Nature 336: 348-352).

The probability of a homologous recombination depends on its isogeny with the genomic DNA of the target cell (te Riele, H., Maandag, E.R. and Berns, A. (1992), Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs, Proc. Natl. Acad. Sci. USA 89: 5128-5132.)

The cloning of chromosomal DNA sections of the locus to be modified, the molecular biological assembly of a bacterial plasmid vector with the  corresponding chromosomal DNA, the targeted integration of the positive selection cassette and equipping both homologous fragments with negative selection cassettes inevitably that the construction of a homologous recombination vector mostly increases a lengthy and technically complex undertaking. It proves to be time-limited thereby the need for inserting the positive selection cassette into the target DNA for unique restriction enzyme recognition sequences (restriction interfaces) to be instructed. This results in complex, time-consuming and labor-intensive cloning. In the targeted mutagenesis of a eukaryotic genome using homologous Recombination therefore often represents the construction of the recombination vector time-limiting step. In the previously known systems that are based on use homologous recombination in yeast or in bacteria are used to construct the Recombination vector required for several weeks. So far there is no experimental System that completes a vector for homologous recombination in short Time allows.

The invention is therefore based on the object to provide a system which Construction of a vector suitable for homologous recombination without complex Cloning steps enabled.

Another object of the invention is to provide a cloning system, which is not unique for inserting the positive selection cassette Restriction enzyme recognition sequences is instructed.

The object is achieved by the subject matter of the claims.

The invention therefore relates to a cloning system for homologous recombination in eukaryotic cells, consisting of a vector adapter system, comprising a Lambda vector with at least two negative selection cassettes, at least two in loxP sites with the same orientation, one between the loxP sites Origin of replication, a resistance marker for bacteria and a genomic stuffer Fragment flanked by a 5 'and 3' restriction interface linker and four different adapter oligonucleotides A, B, C and D, the 5 'nucleotide sequence of the Adapter oligonucleotides A, B, C and D have an end compatible with the restriction enzyme Sfi  contains and the 3 'nucleotide sequence of the adapter oligonucleotides A, B, C, and D to target gene is homologous.

In a preferred embodiment, the 5 'restriction interface linker comprises the Restriction sites 5'-BamHI-Sfi-C, SalI, Sfi-A, BamHI and the 3'- Restriction sites linker the restriction sites 3'-BamHI, Sfi-B, SalI, Sfi-D, BamHI.

A cloning system is particularly preferred, the 5 'nucleotide sequence of the Adapter oligonucleotides A and C the restriction sites SfiA and SfiC and the 5'- Nucleotide sequence of the adapter oligonucleotides B and D, the restriction sites SfiB and SfiD includes.

Surprisingly, the construction is achieved by the cloning system according to the invention of a positive / negative recombination vector is greatly shortened and simplified. At Using the cloning system according to the invention, the cloning time is three Shortened days and enabled the processing of several projects in one time unit. On Previously complex process is thus significantly shortened and cheaper.

The cloning system according to the invention consists of a vector adapter system allows any DNA to be completed by a single cloning step Convert recombination vector with flanking negative selection cassettes.

A preferred cloning system according to the invention comprises a newly constructed lambda vector (Lambda-KO-Sfi), as shown in Fig. 1.

The adapter oligonucleotides used in the cloning system according to the invention enable the efficient amplification of the genomic sequence to be mutated (hereinafter also referred to as the right and left homology arm).

The adapter oligonucleotides are designed so that 15-50, preferably 20-25 base pairs (bp) the 3 'nucleotide sequence is homologous to the respective target gene and the 5' Nucleotide sequence contains an end compatible with the restriction enzyme Sfi. The  Restriction enzyme Sfi enables the construction of several independent interfaces due to the freely selectable middle three base pairs of its restriction recognition sequence.

To amplify the homology arms for the right arm Adapter oligonucleotides with the restriction sites SfiB and SfiD and for the left Arm adapter oligonucleotides with the restriction sites SfiA and SfiC defined. Accordingly, the right homology arm with the adapter oligos B and D and the left Low homology amplified with adapter oligonucleotides A and C. The amplification is carried out using a polymerase with 3'-5 'exonuclease activity, so that the Probability of incorrectly installed bases is minimized.

The size of the PCR products and thus the length of the homology can be freely selected. The two PCR products are used in a ligation approach with the positive selection cassette, which is the Restriction interfaces SfiA and SfiB owns, as well as with the Lambda-KO-Sfi framework that which contains restriction sites SfiC and SfiD. So there are only one Restriction enzyme four incompatible restriction sites were constructed, whereby the ligation of the four fragments can only be done in one way and the cloning runs without false ligation products that otherwise lead to background. Furthermore the invention takes advantage of the high cloning capacity of lambda phage.

The PCR products of the homology arms can be cloned even if only the smallest Amounts have been synthesized. This is a complex test of Ideal conditions for PCR reactions with proof reading polymerases avoided. The Cloning of the recombination vector is independent of that in the genomic DNA existing restriction interfaces, this allows the positive selection cassette to each to place anywhere in the genome and also to set larger deletions. After receiving the adapter oligonucleotide, the recombination vector is completed in three days.

Detailed description of the invention The Lambda vector KO-Sfi

The lambda vector KO-Sfi (see FIG. 1) is derived from the lambda vector KO (Nehls et al. (1994), Biotechniques 17, 4: 770-775). The lambda vector KO is based on the vector Lambda 2001 (Ausubel et al., (1994), Current Protocols in Molecular Biology, John Wiley & Sons, New York). Each genomic fragment inserted into the vector is automatically flanked by two negative selection cassettes, for example HSV-tk. The vector also contains two loxP sites with the nucleotide sequence ATAACTTCGTATAGCATACATTATACGAAGTTAT oriented in the same orientation. After infection of the phage with a bacterial strain expressing cre recombinase (such as BNN132), these loxP sites enable the phage to be converted into a plasmid. Propagation of the plasmid is made possible by an origin of replication and resistance markers for bacteria located within the loxP sites, such as pBluescript (Stratagene, order number # 211201).

In the construction of the lambda vector, a genomic fragment of the phage, preferably of approximately 5 Kb (also referred to here as a stuffer fragment) was placed between the two thymidine kinase genes (HSV-tk) in order to ensure the propagation of the phage. A restriction interface linker with the following structure was used (see also FIG. 1):
5'-BamHI, Sfi-C, SalI, Sfi-A, BamHI
3'-BamHI, Sfi-B, SalI, Sfi-D, BamHI

The Sfi interfaces have the following sequences:
Sfi A: ggcentagnggcc
Sfi B: ggccngcgnggcc
Sfi C: ggccnctgnggcc
Sfi D: ggccncagnggcc

In addition, two sequencing primers specific to the respective ends were derived from the above-mentioned newly introduced linker sequences:
MM-D 5'-gacggatcctcggccactg
MM-C 5'-ctcgaggatccggccactg

For use as a cloning vector, the Lambdaphagen DNA is first with Sfi hydrolytically split. The fragments obtained are purified and in a second  Cut digestion with the restriction enzyme SalI. That should end each three times be cut and religation products avoided during cloning. The Lambda Arms are then cleaned by sucrose gradient centrifugation and, in this way, by the stuffer fragment. The PCR products are ligated at Standard conditions and in vitro packaging are done using one of Stratagene manufactured packaging extract (Gigapack plus).

The adapter oligonucleotides (see also FIG. 1)

The adapter oligonucleotides are used to amplify the homology arms (0.8-12 kb) mutating genomic sequence and to incorporate these amplified sequences in the Cloning vector Lambda-KO-Sfi.

The 5 'ends of the adapter oligonucleotides A, B, C and D each define the four different Sfi interfaces. After amplification of the genomic sequences, ie the right and left homology arms, and purification of the two PCR products, these are hydrolytically cleaved with Sfi. In order to ensure complete cleavage, an additional three nucleotides were placed at the 5 'ends of the adapter oligonucleotides (underlined in the table below). At the 3 'end of the adapter oligonucleotides there is a nucleotide sequence homologous to the nucleotide sequence of the respective candidate gene (shown in the following list as (N _genomic ) 25).
Sfi-A: cga ggc cgc tat ggc c (N _genomic ) 25
Sfi-B: gac ggc cag cga ggc c (N _genomic ) 25
Sfi-C: cag ggc cac tgc ggc c (N _genomic ) 25
Sfi-D: cag ggc cac tgc ggc c (N _genomic ) 25

The selection cassette

In order to simultaneously knockout a gene, expression of the endogenous gene To monitor, an IRES-β-galactosidase-MCS-neomycin cassette with the Sfi interfaces A and B provided and in the LambdaKO-Sfi vector as a stuffer fragment cloned in. In this way, the expression of the endogenous gene can be monitored at the same time  become. With a lambda phage DNA preparation, DNA also falls Selection cassette in molar ratio, which can be cleaned at the same time.

The abbreviations used have the following meaning according to the invention.
In addition to the abbreviations used in Duden, the usual codes for amino acids and nucleotides as well as the common abbreviations for restriction enzymes, polymerases, etc., the following abbreviations were used:
Bp: base pair
ES cells: embryonic stem cells
hprt gene: hypoxanthine phosphoribosyl transferase gene
HSK: Herpes Simplex Virus
MCS: multiple cloning site
µl: microliter
Ng: nanogram
Ori: Origin of replication PCR: polymerase chain reaction
tk gene: thymidine kinase gene
U: unit

The invention is further illustrated by the following drawing:

Fig. 1A is a graphical representation of a cloning system according to the invention, consisting of a lambda vector (Lambda-KO-Sfi) and four Adapteroligonukleotiden A, B, C and D.

The Lambda KO-Sfi: The LambdaKo-Sfi vector consists of the right and left arm of the Lambda2001 (Ausubel, F.M., Brent, R., Klingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (1994), Current Protocols in Molecular Biology, John Wiley & Sons, New York) by Nehls et al. was changed (Nehls et al. (1994), Biotechniques 17, 4: 770-775): adjacent to the Lambda arms were two in the same Orientation-oriented loxP points introduced. Between the loxP positions in the  right arm of the lambda the gene for β-lactamase and an origin of replication (ori) advertises. A genomic stuffer fragment is on both sides of two cassettes for the negative Selection flanked. (Mansour, S.L., Thomas, K.R. and Capechi, M.R. (1988), Disruption of the proto-oncogene int-2 in mouse embryo derived stem cells: a general strategy for targeting mutations to non-selectable genes, Nature 336: 348-352). At the 5 'end of the stuffer The following restriction interface linker was newly introduced: BamHI, Sfi-C, SalI, Sfi-A, BamHI and a second restriction site linker to the 3 'end of the stuffer Fragments: BamHI, Sfi-B, SalI, Sfi-D, BamHI. The Sfi interfaces are as follows defined: Sfi-A: ggccNTAGNggcc; Sfi-B: ggccNGCGNggcc; Sfi-C: ggccNCTGNggcc; Sfi-D: ggccNCAGNggcc.

FIG. 1B shows a homologous recombination vector for targeted mutagenesis of ES cells, the cut from the purified Sfi lambda KO-Sfi arms consists, in which the PCR products (right and left homology arm) and ES cell selection cassette in were ligated in a ligation step. For this purpose, the left homology arm was amplified with adapter oligonucleotides C and A, the right arm with adapter oligonucleotides B and D. The total size of the two PCR products can be freely selected within the limits of 0.8-12 kb.

Examples

The following examples illustrate the invention, but are not limitative understand.

example 1 Construction of a homologous recombination vector for the Hprt gene

To construct a homologous recombination vector for the Hprt gene, the carried out the following experimental steps:

A. Select genomic olisonucleotides for PCR of the homology arms

In the selected example, exon III of the Hprt gene is deleted. This mutation leads to Loss of function of Hprt, as already described by Hasty et al. (1991) Mol. Cell. Biol. 11: 5586-5591, described.

The left homology arm of Hprt is 3639 bp long and ends with the 17th base of the 3rd exon. The right homology arm, which is 950 bp long, begins in the 3rd intron with the 39th base. The bases in bold in the following list identify the adapter sequences.

B. Genomic PCR

The PCR was carried out with the following reagents:

The denaturation was carried out for 45 seconds at 94 ° C., the oligonucleotides were attached ("Annealing") for 1 minute at 56 ° C, the synthesis for 3 minutes at 72 ° C. This cycle was repeated 34 times.

C. Purification of the PCR products

The PCR products were prepared using the QIAquick PCR Purification Kit (Order number # 21806) cleaned according to the manufacturer's descriptions. The Elution volume was 30 µl.

D. Sfi digestion of the PCR products

The purified PCR product was mixed with 40 U Sfi (Biolabs, order number # 123L) digested for three hours at 50 ° C and then again using the QIAquick PCR Purification Kit purified and eluted from the column in 30 µl.

E: Ligation of the Sfi-cut PCR products with Lambda-KO-Sfi and the positive selection cassette

A typical ligation batch consisted of the following amounts in a 10 µl batch:
50 ng Lambda KO Sfi arms (Sfi cut)
10 ng selection cassette (Sfi cut)
1 ng right homology arm
1 ng left homology arm
1 × ligation buffer (Boehringer)
1 U T4 ligase (Boehringer, order number # 716354)

The ligation was carried out at room temperature for two hours.

F. In vitro packaging

2 ul of the above-mentioned ligation batch were for 1.5 hours at room temperature packed with 10 µl packaging extract in vitro (Stratagene, Gigapack III plus, order number # 200204).

G. Library plating

10 and 50 µl of the packaging reaction were used for infection with 300 µl of a growing one Culture of C600 bacteria (Stratagene, order number # 200261) and used Lambda plates (Current Protocol in Molecular Biology) incubated overnight.

H. Plasmid conversion

Individual plaques were placed in 500 ul SM phage buffer (Ausubel, F.M. et al., (1994) Current Protocol in Molecular Biology, John Wiley & Sons, New York). 50 µl each Phages in SM phage buffer were infected with 100 ul of a growing BNN 132 culture (30 minutes, 37 ° C) and then a liquid culture TB medium (Ausubel, F.M. et al., (1994) Current Protocol in Molecular Biology John Wiley & Sons, New York) plus 100 µg Ampicillin / ml (Amersham, order number # US11259-25) inoculated and overnight at 30 ° C incubated in the bacterial shaker.

I. Plasmid preparation

The plasmid DNA was analyzed by QIAgen column chromatography (Qiagen, order number # 12143) according to the manufacturer's instructions.  

SEQUENCE LOG

Claims (7)

1. Cloning system for homologous recombination in eukaryotic cells, consisting of a vector adapter system comprising a lambda vector, characterized in that this

• a) at least two negative selection cassettes;
• b) at least two loxP sites with the same orientation;
• c) an origin of replication located between the loxP sites;
• d) a resistance marker for bacteria; and
• e) contains a genomic stuffer fragment flanked by a 5 'and 3' restriction site linker;

and four different adapter oligonucleotides A, B, C and D, the 5 'nucleotide sequence of adapter oligonucleotides A, B, C and D containing an end compatible with the restriction enzyme Sfi and the 3' nucleotide sequence of adapter oligonucleotides A, B, C and D for target gene is homologous. 2. Cloning system according to claim 1, wherein the 5'- Restriction sites linker the restriction sites 5'-BamHI-Sfi-C, SalI, Sfi-A, BamHI and the 3'-restriction site linker the restriction sites 3'-BamHI, Sfl- B, SalI, Sfi-D, BamHI. 3. Cloning system according to claim 1, wherein
the 5 'nucleotide sequence of the adapter oligonucleotides A, the restriction sites SfiA,
the 5 'nucleotide sequence of the adapter oligonucleotides C the restriction sites SfiC,
the 5 'nucleotide sequence of the adapter oligonucleotides B, the restriction sites SfiB, and
the 5'-nucleotide sequence of the adapter oligonucleotides D comprises the restriction sites SfiD. 4. Cloning system according to claim 1, wherein the to the respective target gene homologous 3 'nucleotide sequence of adapter nucleotides A, B, C, and D 15 to 50 bp, preferably comprises 20-25 bp. 5. A method for producing a homologous recombination vector consisting of
a lambda vector,
at least two negative selection cassettes,
at least one positive selection cassette and
a left and right homology arm;
comprising the steps:

• a) selecting sequences homologous to the target gene (genomic oligonucleotides) for the amplification of the 5 'and 3' homology arms by means of PCR;
• b) amplification of the 5 'and 3' homology arms by means of PCR and purification of the PCR products;
• c) restriction digestion of the purified PCR products using restriction enzyme Sfi;
• d) ligation of the Sfi-cut fragments with an Sfi-cut lambda vector and an Sfi-cut positive selection cassette;
• e) in vitro packaging of the ligation products and plating of the phage library; and
• f) plasmid conversion and extraction of the DNA of the homologous recombination vector.

6. A method for producing a homologous recombination vector according to Claim 5, characterized in that the lambda vector is lambda KO-Sfi. 7. Use of the cloning system according to one of claims 1 to 4 to in eukaryotic cells, especially embryonic stem cells, defined the genome Change loci.