Patent Application: US-44056695-A

Abstract:
an improved method of gene targeting , referred to as pcr - based gene targeting is disclosed , which generates cell lines or 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 is especially applied to murine macrophage cytokine - inducible nitric oxide synthase .

Description:
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 understood from the following detailed description of preferred embodiments of the invention taken in conjunction with the appended drawings . fig1 shows : mapping the murine nos2 genomic locus using long - range pcr . approximate genomic distance between exons was estimated for primer pairs that generated an amplicon ( lanes 1 , 5 , 6 , 7 , 8 , and 9 ). see table 1 and methods below for primer sequences , putative exon locations , and size of amplicons . the largest amplicon is about 7 kb and includes putative exons 4 to 7 ( lane 8 ). this amplicon was used to construct a gene targeting vector . size markers are shown in lane m . fig2 in two parts , namely , fig2 a ( panel a ) and fig2 b ( panel b ) shows : validation of pcr - based mapping using in - gel southern blot analysis where genomic dna fragment sizes are the same as those estimated by pcr analysis . ( panel a ) murine genomic dna ( 8 μg / lane ) was digested with ecori ( e ), hindiii ( h ), or saci ( s ). ( panel b ) dna was double - digested with ecori and hindiii ( eh ), or ecori and saci ( es ), or hindii and saci ( hs ). samples were electrophoresed within the same gel and hybridized with probe a ( fig3 ). dots represent size markers as described in fig1 . fig3 shows : schematic diagram of pcr - based gene targeting of the murine nos2 locus . ( exons , genomic locus ) genomic positions of putative exons were determined by the pcr strategy shown in fig1 . distance between exons 2 to 4 is represented by interrupted lines . the distance between the second and third hindiii ( h ) sites was determined by southern blot analysis to be approximately 20 kb . other restriction sites are clai ( c ), ecori ( e ), noti ( n ), saci ( s ), and xbai ( x ). for the sake of clarity , only relevant restriction sites are shown . ( pcr amplicon ) primers 1 and 2 ( table 1 , gel lane 8 , primer pairs ) are represented by arrow heads and were used to generate a 7 kb amplicon , represented by the shaded bar . this was constructed by cloning the 7 kb amplicon into a bluescript vector containing the pgk - tk selection cassette and inserting the pkg - neo selection cassette into the saci site of exon 6 . the targeting vector was linearized with noti and transfected into es cells . the murine nos2 locus after homologous recombination . primers 3 and 4 ( arrow heads ) were used to screen for homologous recombination involving the short - arm . homologous recombination was confirmed by southern blot analysis using the 0 . 3 kb genomic probe a and the 1 . 7 kb pgk - neo probe b . fig4 in three parts , namely fig4 a ( panel a ), fig4 b ( panel b , and fig4 c ( panel c ) shows : homologous recombination was detected using southern blot analysis of genomic dna from transfected es cell clones , hybridized with probe a . genomic dna was isolated from es cell clones roh . 1 ( lane a ), roh . 2 ( lane b ), and e14 . 1 ( lane c ). ten micrograms of dna was digested with ecori ( panel a ), ecori and hindiii ( panel b ), or hindiii ( panel c ). the band representing the allele that has undergone homologous recombination is marked with an asterisk . hybridizations were performed simultaneously , using the same probe preparation . fig5 in two parts , namely fig5 a ( panel a ) and fig5 b ( panel b ) shows : confirmation of homologous recombination using probe b . genomic dna was isolated from es cell clones roh . 2 ( lane a ), roh . 1 ( lane b ), and e14 . 1 ( lane c ). ten micrograms of dna was digested with ecori ( panel a ) and hindiii ( panel b ). the band representing the allele that has undergone homologous recombination is marked with an asterisk . hybridizations were performed as in fig4 . in order to illustrate the invention in further detail , the following specific laboratory examples were carried out with the results indicated . although specific examples are thus illustrated , it will be understood that the invention is not limited to these specific examples or the details therein . pcr . the genomic organization of the human nos2 locus ( 24 ) and sequences of human ( 25 ) and murine nos2 cdna &# 39 ; s ( 26 - 28 ) were used as a basis for approximating where putative exon boundaries might be . oligonucleotide primers were obtained that bind putative exons 1 to 8 , 12 , 18 , and 23 ( protein chemistry laboratory , table 1 , fig1 ). genomic dna from the raw 264 . 7 murine macrophage cell line ( h - 2 d , atcc tib 71 ) ( 29 ) was used as a template . conditions for pcr : ( 93 ° c ./ 5 min ; 93 ° c ./ 1 min , 68 ° c ./ 10 min ) for 16 cycles ; ( 93 ° c ./ 1 min , 68 ° c ./ 10 min plus 15 sec ) for 12 cycles ; the geneamp xl pcr kit ( perkin elmer ) which contains a mixture of thermostable dna polymerases ( rtth and vent ®) was used . additional primers were as follows : sense — 5 ′ acac tacata ctttatgccaccaacaatggcaac 3 ′ [ seq id no : 13 ] and anti - sense — 5 ′ ggagataggacatagttcaacatctcc 3 ′ [ seq id no : 15 ] primers in putative exons 7 and 12 , respectively . sense primer of lane 10 and anti - sense primer — 5 ′ ggatgctgctgagggctctgttgagg 3 ′ [ seq id no : 16 ], in putative exons 7 and 18 . sense primer of lane 10 and anti - sense primer — 5 ′ ctcagggagctggaagccactgacacttcg 3 ′ [ seq id no : 17 ], in putative exons 7 and 23 . plasmid construction . a 7 . 0 kb amplicon spanning putative exons 4 to 7 was subcloned into the bluescript skii + plasmid ( stratagene ), using noti and clai restriction sites that were added to the primers ( 30 , 31 ). pcr - derived insert was mapped using restriction endonucleases and partially sequenced using the di - deoxy sequencing method ( 32 ) and a sequenase t7 dna sequencing system ( amersham ). exon 6 was opened using a partial saci digest and the 1 . 7 kb phosphoglycerokinase ( pgk )- neo cassette inserted ( 33 - 35 ). a 2 . 6 kb pgk - tk cassette was inserted 5 ′ of exon 4 ( 33 ). the long - arm spanned exons 4 to 6 ( 5 . 9 kb ) and the short - arm spanned exons 6 to 7 ( 1 . 1 kb ). this targeting vector was linearized at the noti site . cells . male e14 es cells ( 36 ) were co - cultured on geneticin ( g418 ) antibiotic - resistant murine embryonic fibroblasts ( 36 - 38 ) in high glucose dmem nutrient culture medium ( 4 . 5 g / l ; gibco ) supplemented with 15 % fetal calf serum ( hyclone ), glutamine ( 2 mm ), non - essential amino acids ( 0 . 1 mm ), penicillin ( 100 u / ml ), streptomycin ( 100 μg / ml ), 2 - mercaptoethanol ( 50 μm ), and leukemia inhibitory factor ( 1000 u / ml , gibco ) at 6 % co 2 . transfection and selection . es cells ( 2 × 10 7 cells ) were electroporated using the btx 600 electroporation system ( 340 v , 100 μf , 0 . 4 cm gap ) and 50 μg of noti - linearized targeting vector . electroporated cells were plated onto 10 cm dishes and selection initiated 24 hr later using 250 μg / ml g418 ( gibco ) and 2 μm ganciclovir ( syntex ). colonies were picked on day 6 . screening . genomic dna was isolated from es clones and initially evaluated using pcr analysis . primer 3 , which is external to the short - arm but within exon 7 ( 5 ′ gttgccattgttggtggcataaag 3 ′) [ seq . id no : 12 ] and primer 4 , which is specific for the pgk - neo cassette ( 5 ′ cggtagaattatcgaattcctgcagc 3 ′) [ seq . id no : 18 ] were used . accurate targeting events were determined by southern blot analysis , using a 0 . 3 kd bamhi / ncoi genomic probe located external to the short - arm ( probe a , fig3 ), and a 1 . 7 kb pgk - neo probe ( probe b , fig3 ). genomic dna was digested with ecori and / or hindiii , size - fractionated on a 0 . 7 % agarose gel which was dried , pre - hybridized , hybridized using conventional techniques for in - gel southern blot analysis ( 39 - 41 ). gels were washed at 68 ° c . in 4 × ssc / 0 . 1 % sds followed by 0 . 1 × ssc / 0 . 1 % sds for 1 hr . auto - radiograms were digitized using an hp scanjet iic / adf scanner ( hewlett packard ), a powermac 7100 / 66 , and deskscan ii software ( hewlett packard ). to determine where a pgk - neo selectable marker cassette could be placed in order to disrupt the nos2 gene , the genomic organization of the murine locus was initially investigated . a combination of southern blot analysis and long - range pcr ( 23 ) was used to map genomic regions of interest . using the organization of the human nos2 gene ( 24 ) the location of exons in murine cdna sequence ( 26 ) was predicted ( 26 ). the distance between putative exons was then mapped using genomic dna template isolated from the raw 264 . 7 murine macrophage cell line . long - range pcr was performed using primer - pairs listed in table 1 and fig1 . geneamp xl , a blend of thermostable dna polymerases , was used to generate amplicons containing an intron flanked by a portion of exons containing primer - binding sites ( fig1 ). the distance between exons 1 and 2 of the murine nos2 gene is similar to that reported for the human locus ( 24 ). they are approximately 1 . 6 and 1 . 8 kb , respectively ( lane 1 , fig1 ). distances between exons 2 , 3 , and 4 of the human nos2 gene are approximately 1 . 1 , 0 . 9 , and 2 . 0 kb , respectively . an amplicon using primers specific for putative exons 2 to 4 ( lanes 2 , 3 , and 4 ; fig1 ) could not be generated , although a 20 kb amplicon using control plasmid and primers provided with the geneamp xl kit was generated . the organization of exons 4 through 7 appears to be comparable between human and murine nos2 genes . the distances are approximately 1 . 2 , 6 . 5 , and 1 . 7 kb in the human nos2 gene and are approximately 1 . 8 , 4 . 1 , and 1 . 1 kb in the murine gene ( lanes 5 , 6 , and 7 ; fig1 ). to determine the capacity of this pcr system for generating genomic amplicons larger than 7 kb ( lane 8 , fig1 ), primer - pairs from putative murine exons 7 , 12 , 18 and 23 were used because inter - exon distances are approximately 7 . 5 , 15 , and 22 kb in the human nos2 gene ( 24 ). that genomic amplicons were not obtained for this region of the murine nos2 locus suggests the presence of differences in organization or the location of intron - exon boundaries compared to the human gene ( lanes 10 , 11 , and 12 ; fig1 ). pcr - based genomic mapping was further validated by comparing pcr - generated data about inter - exon distances to data obtained by southern blot analysis ( fig2 ). murine genomic dna was digested with ecori , hindiii or saci and probed with a 0 . 3 kb dna fragment used to screen es cells ( probe a , fig3 ). this probe was derived from a 1 . 3 kb amplicon spanning putative exons 7 and 8 ( lane 9 , fig1 ). probe a detected approximately 8 . 5 kb ecori , 20 kb hindiii , and 4 . 0 kb saci fragments on southern blot analysis ( panel a , fig2 ). the 8 . 5 kb distance predicted by pcr - mapping between the two ecori sites flanking putative exon 6 is the same length of the fragment detected by probe a on southern blot analysis . further mapping of the region around probe a was done by performing double digests of murine genomic dna using ecori and hindiii , ecori and saci , or hindiii and saci . the pcr - generated map predicts an approximately 5 . 6 kb ecori / hindiii , and a 2 . 2 kb ecori / saci fragment . these fragment sizes are the same as those determined by southern blot analysis . for pcr - based gene targeting , a 7 kb amplicon flanked by portions of exons 4 and 7 ( lane 8 , fig1 ) was generated . the pgk - neo positive selectable marker gene was inserted into putative exon 6 of this amplicon , at a saci site ( fig3 ). this approach resulted in a 1 . 1 kb short - arm and a 5 . 9 kb long - arm of genomic dna sequence homologous to the murine nos2 chromosomal locus . the pgk - tk negative - selectable marker gene was placed 5 ′ of exon 4 . exon 6 of the nos2 gene was targeted for the following reasons : the ability to generate a 7 kb amplicon between exons 4 and 7 using existing primer pairs initially prompted investigation of exons located within this region . there is presently no evidence for alternative splicing of murine nos2 mrna between exons 2 and 7 ( 6 , 7 , 42 ). the binding domains for calmodulin ( 24 ), tetrahydro - biopterin ( 43 ), the flavin nucleotides fmn and fad ( 24 ), and nadph ( 44 ) are all 3 ′ of exon 6 . part of the heme binding site appears to be encoded by exon 6 ( 45 ). thus , if disruption of the murine nos2 gene at exon 6 results in a stable truncated protein , it should be non - functional . pcr - based targeting vector linearized with noti was then transfected into e14 es cells using the electroporation method . following a 24 hr recovery period , cells were selected in media containing g418 and ganciclovir ( ganc ). colonies resistant to g418 and ganc were picked , expanded , and screened from homologous recombinants by pcr analysis using a primer - pair specific for a region 3 ′ of the short - arm of the targeting vector and for the pgk - neo cassette ( table 1 , fig3 ). clones found to be positive by pcr analysis were confirmed by southern blot analysis using probes a and b ( fig3 ). in addition to the germline 8 . 5 kb ecori fragment , a new 2 . 4 kb ecori fragment was detected by probe a when targeting vector and chromosomal dna undergo homologous recombination ( table 2 , lane a in fig4 a and 4 b ). double - digestion with ecori and hindiii resulted in a 5 . 6 kb germline fragment , whereas the 2 . 4 kb ecori new fragment was unchanged ( table 2 , fig4 b ). digestion with hindiii generated an approximately 20 kb germline fragment which is difficult to resolve from the 19 kb fragment of the targeted allele ( table 2 , fig4 c ). probe b detects the transfected pgk - neo cassette and fragments of the endogenous pgk gene ( table 2 ). a 4 . 4 kb ecori and an approximately 4 . 3 kb hindiii fragment represent the endogenous pgk gene ( table 2 , fig5 ). dna from a correctly targeted clone , roh . 1 , has the expected 8 kb ecori and approximately 19 kb hindiii fragments representing the targeted allele ( table 2 , lane a of fig5 ). in contrast , the results of probing dna from clone roh . 2 with probe b are consistent with a random insertion of the targeting construct . approximately 4 . 3 kb ecori and 2 . 3 kb hindiii fragments are detectable by probe b ( fig5 a and 5 b ). no other bands were observed , suggesting that no additional copies of the targeting vector were integrated into the genome of these es cell clones . the targeting frequency of pcr - based gene targeting is of similar order - of - magnitude as conventional gene targeting ( 2 , 46 ). in the first 41 double - resistant clones screened , four correctly targeted clones ( table 3 ) were identified . thus , approximately 1 of 10 double - resistant clones were accurately targeted using the pcr - based gene targeting method of the invention . various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the invention . it is intended that all such other examples be included within the scope of the claims appended hereto . 3 . bronson , s . k . & amp ; smithies , o . 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