Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1995-05-16
2003-07-01
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S091200, C435S354000
Reexamination Certificate
active
06586250
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to an improved method of gene targeting that is more rapid and less costly than conventional gene targeting. More particularly, the invention relates to a PCR-based gene targeting method to develop cell lines and mice in which at least one allele of a specific gene is disrupted by double homologous recombination of a PCR-derived targeting vector with chromosomal DNA. The method of the invention is especially applied to murine macrophage cytokine-inducible nitric oxide synthase (Mø-iNOS).
PCR (polymerase chain reaction) is an in vitro method of amplifying DNA sequences. It is an enzymatic process that is carried out in discreet cycles of amplification, each of which can double the amount of target sample. For a brief recent review, see Arnheim and Levenson, “Polymerase Chain Reaction”,
Chem
. &
Eng. News
, Oct. 1, 1990, pp. 36-47. See also U.S. Pat. Nos. 4,683,195 and 4,683,202.
Long-range PCR is an in vitro method of amplifying longer DNA sequences using a combination of thermostable DNA polymerases. See Cheng et al.,
Proc. Natl. Acad. Sci. USA
91, 5695-5699 (1994).
Nitric Oxide (NO) has been shown to modulate vascular tone, smooth muscle cell proliferation, platelet aggregation and adhesion, leukocyte adhesion, and macrophage (Mø) cell-mediated cytotoxicity. Thus, NO appears to be medically important in vascular biology and immunology. For a recent brief review, see Feldman, Griffith and Stuehr, “The Surprising Life of Nitric Oxide”,
Chem
. &
Eng. News
, Dec. 20, 1993, pp. 26-38.
NO is produced from L-arginine by two classes of NO synthase (NOS). A constitutive NOS (cNOS) is rapidly activated for a very brief duration by an elevation in intracellular Ca
2+
and calmodulin binding. The activity of cytokine-inducible NOS (iNOS) is slowly increased, due to transcriptional induction, and sustained for days.
The gene for Mø-iNOS has been cloned and shown to be a distinct member of the NOS gene family. Consequently, the iNOS isoform is an attractive potential target for pharmacological modification of inflammation.
(Note: Literature references on the following background information and on conventional test methods and laboratory procedures well known to the ordinary person skilled in the art, and other such state-of-the-art techniques as used herein, are indicated in parentheses, and appended at the end of the specification.)
Gene targeting has been used heretofore to develop animal models of disease, explore mechanisms of biological development, and assess the functional importance of molecules. The subject of gene targeting has been reviewed recently (1-8). This technique makes use of double homologous recombination in murine embryonic stem (ES) cells between chromosomal DNA and homologous sequences in the targeting vector (9-11). Transfected ES cells are screened and those with a correctly targeted allele are injected into blasto-cysts to generate mice having a defined germline mutation.
Conventional targeting constructs consist of neo (neomycin phosphotransferase) positive-selectable marker gene flanked by a total of at least 4 kb (kilobases) of homologous DNA sequence (11).
The tk negative-selectable marker gene is commonly used in conventional targeting constructs (12-19).
The frequency of homologous recombination varies as a function of insertional- versus replacement-vector design (11), length of DNA homology (11, 20, 21), and degree of polymorphic variation between the vector and the chromosome (22).
In conventional targeting, the targeting vector is derived from genomic DNA fragments obtained using conventional library cloning techniques. Typically, the DNA fragments are cloned from isogenic or non-isogenic murine genomic libraries. This process is time consuming and costly, such that commercial businesses have been formed that specifically offer this service at market prices.
Accordingly, a method of gene targeting that is faster and less costly whereby it bypasses the conventional library cloning process would have significant advantages in the field.
BRIEF DESCRIPTION OF THE INVENTION
In accordance with the present invention, a novel method, referred to as PCR-based gene targeting, is provided. According to this method, large DNA fragments homologous to the gene to be targeted, for example the inducible nitric oxide synthase (NOS2) gene locus, are obtained by using long-range PCR (23).
A large PCR-derived amplicon, for example the 7 kb NOS2 homologous amplicon, is used to construct a targeting vector where the neo gene is flanked by PCR-derived homologous DNA sequences. The vector also includes a thymidine kinase (tk) negative-selectable marker gene.
The resulting targeting vector is then transfected into murine ES cells, where homologous recombination takes place. That is, following transfection into ES cells, the PCR-based targeting vector undergoes efficient homologous recombination into the NOS2 locus.
According to a preferred method of the invention, the PCR-based gene targeting comprises the steps of:
(a) obtaining a PCR-derived homologous genomic DNA fragment by applying long-range PCR to a murine genomic DNA template to generate a large genomic amplicon spanning the exons of a gene locus of interest by employing
(i) a first oligonucleotide primer having a sequence specific for the 5′ end of the target region and a suitable restriction site added at its 5′ end, and
(ii) a second oligonucleotide primer having a sequence specific for the 3′ end of the target region and a suitable restriction site added at its 3′ end, whereby said primers bind to, respectively, the 5′ and 3′ ends of said target area,
(b) producing a PCR-based targeting vector by
(i) inserting the 1.7 kb pgk-neo positive selectable marker gene into said amplicon at a suitable restriction site within said target region and
(ii) inserting the 2.6 kb pgk-tk negative selectable marker gene at the 5′ or 3′ end of said amplicon to linearize said targeting vector, and
(c) thereafter transfecting said PCR-based targeting vector into ES cells whereby said targeting vector undergoes efficient homologous recombination into said gene locus.
By the term “large” genomic amplicon is meant an amplicon of at least 4 kb in length. The large genomic amplicon is exemplified by a 7 kb genomic amplicon spanning exons 4 to 7 of the NOS2 gene locus consisting of a 1.1 kb short-arm spanning exons 6 to 7 and a 5.9 kb long-arm spanning exons 4 to 6 of said NOS2 gene locus.
A preferred restriction site added at the 5′ end of said first oligonucleotide primer is ClaI; a preferred restriction site added at the 3′ end of said second oligonucleotide primer is NotI.
A preferred restriction site within the target region for insertion of the pgk-neo positive selectable marker gene is the SacI site at the 3′ end of exon 6 of the NOS2 amplicon.
A preferred murine genomic DNA template is the genomic DNA from the conventional, well-known RAW 264.7 murine macrophage cell line (H-2
d
) which is readily available from the American Type Culture Collection, Rockville, Md., under Accession Number ATCC TIB 71.
Preferred oligonucleotide primers are shown in the Examples and Table 1, below.
By this method, PCR-based gene targeting is applied to the mouse-inducible nitric oxide synthase locus, whereby ES cell lines are generated in which the NOS2 gene is disrupted on one chromosome.
This method expedites the acquisition of homologous genomic DNA sequences, and simplifies the construction of targeting plasmids by making use of defined cloning sites.
It also provides a substantial time and cost savings for appropriate homologous recombination projects. The PCR-based gene targeting method of the invention thus is a valuable alternative to the conventional cloning approach.
DETAILED DESCRIPTION OF THE INVENTION
While the specification concludes with claims particularly pointing out and distinctly claiming the subject matter regarded as forming the present invention, it is believed that the invention will be better understo
Loeb Bronwen M.
Meyer Scott J.
Washington University
Yucel Remy
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