Effects of noise on inferior colliculus evoked potentials and cochlear anatomy in young and aged chinchillas

Site-specific modification of the chromosomal immunoglobulin locus by gene targeting is a powerful tool in studying the molecular requirements for immunoglobulin gene structure and function and in the production of engineered antibodies. Here, we describe a two step- integration then excision-gene targeting procedure for introducing planned genetic alterations into the chromosomal immunoglobulin locus. The efficiency of gene targeting with an enhancer-trap vector in which an enhancerless neo and HSV-tk gene were inserted into the vector backbone was compared to that of the corresponding enhancer-positive vector. Both insertion vectors also contained homology to the chromosomal immunoglobulin target locus along with the desired genetic alteration. The first step involved insertion of the transferred vector into the target locus by homologous recombination. An approximately 15-fold enrichment in the frequency of vector insertion was obtained with the enhancer-trap compared to the enhancer-positive vector. The majority of targeted cells (75%) contained a single copy of the vector integrated into the chromosomal immunoglobulin locus. The second step involved excision of the integrated vector by intrachromosomal homologous recombination between the duplicated region of homology that removed the integrated vector, neo and tk genes along with one copy of homologous DNA. Vector excision was very efficient generating G418S, FIAU(R) secondary recombinants at the high rate of approximately 10(-3)/cell generation. In the secondary recombinants, the overall structure of the chromosomal immunoglobulin locus was restored with the desired genetic alteration being present in an expected proportion of the cells.     

Targeted recombination at the Chinese hamster APRT locus using insertion versus replacement vectors

In this study, we have examined the effects of targeting vector configuration and site of vector linearization on the frequency of targeted recombination at the endogenous CHO APRT locus, and have analyzed the types and class distributions of APRT+ recombinants obtained in APRT targeting experiments employing uncut circular, insertion-type (ends-in), and replacement-type (ends-out) configurations of the same pAG7 targeting vector, including configurations produced by introduction of a double-strand break (DSB) at sites either within, or at the 5' or 3' boundaries of APRT targeting homology. Our results suggest that: 1) plasmid-chromosome targeted recombination in mammalian cells may not be stimulated to the same degree by a DSB in the targeting vector as by a DSB in the chromosomal target; 2) recombinant class distributions are highly dependent upon targeting vector configuration; and 3) one-sided invasion mechanisms may play a significant role in homologous recombination in mammalian cells.     

An in vivo assay for measuring the recombination potential between DNA sequences in mammalian cells

Mammalian intermolecular recombination vectors that place the recombination junction within the intron of a selectable marker gene are presented. Many of the previously reported recombination assays require that recombination occur homologously and that they occur within the coding region of the selectable marker. This vector system involves the use of a human thymidine kinase (tk) minigene and measures the recombination frequency between any chosen DNA sequences, in mammalian thymidine kinase negative cells. The tk minigene is divided into a 5' vector and a 3' vector. In the 5' vector, the DNA sequence of interest is inserted in the proximal portion of tk intron 2. In the 3' vector, the DNA sequence of interest is inserted in the intron sequence between the proteolipid protein exon 2 and tk exons 3-7. Recombination through the DNA sequences of interest, either homologous or illegitimate, will reconstruct a functional tk minigene. The recombination junction is spliced out of the transcribed mRNA and thymidine kinase positive cells can be selected in hypoxanthine-aminopterin-thymidine medium. We have tested these vectors to measure the recombination potential of two Alu repetitive sequences (BLUR 8 and BLUR 11) against a control DNA sequence. BLUR 8 and BLUR 11 do not seem to recombine at a significantly higher frequency over that of the control DNA sequence. These recombination vectors display similar sensitivity to previous recombination systems, but allow tremendous flexibility in the choice of potentially recombinogenic sequences.     

Processing of DNA prior to illegitimate recombination in mouse cells

In mammalian cells, the predominant pathway of chromosomal integration of exogenous DNA is random or illegitimate recombination; integration by homologous recombination is infrequent. Homologous recombination is initiated at double-strand DNA breaks which have been acted on by single-strand exonuclease. To further characterize the relationship between illegitimate and homologous recombination, we have investigated whether illegitimate recombination is also preceded by exonuclease digestion. Heteroduplex DNAs which included strand-specific restriction markers at each of four positions were generated. These DNAs were introduced into mouse embryonic stem cells, and stably transformed clones were isolated and analyzed to determine whether there was any strand bias in the retention of restriction markers with respect to their positions. Some of the mismatches appear to have been resolved by mismatch repair. Very significant strand bias was observed in the retention of restriction markers, and there was polarity of marker retention between adjacent positions. We conclude that DNA is frequently subjected to 5'-->3' exonuclease digestion prior to integration by illegitimate recombination and that the length of DNA removed by exonuclease digestion can be extensive. We also provide evidence which suggests that frequent but less extensive 3'-->5' exonuclease processing also occurs.     

The structures of integration sites in transgenic rice

Extensive genomic sequencing and sequence motif analysis have been conducted over the integration sites of two transgenic rice plants, #478 and #559, carrying the luciferase gene and/or hygromycin phosphotransferase gene. The transgenes reside in a region with inverted structure and a large duplication of rice genome over 2 kb. Integration was found at the AT-rich region and/or at the repetitive sequence region, including a SAR-like structure, retrotransposon and telomere repeats. The presence of a patch of sequence homology between plasmid and target DNA, and a small region of duplication involving the target DNA around the recombination site, implicated illegitimate recombination in the process of gene integration. Massive rearrangement of genomic DNA including deletion or translocation was also observed at the integration site and the flanking region of the transgene. The recognition sites of DNA topoisomerases I or II were observed in the rearranged sequences. Since only three junctions of transgenic rice were implicated in the illegitimate recombination and extensive rearrangement of the rice genome, rice protoplasts may be active in this process.     

Stimulation of intrachromosomal homologous recombination in human cells by electroporation with site-specific endonucleases

In somatic mammalian cells, homologous recombination is a rare event. To study the effects of chromosomal breaks on frequency of homologous recombination, site-specific endonucleases were introduced into human cells by electroporation. Cell lines with a partial duplication within the HPRT (hypoxanthine phosphoribosyltransferase) gene were created through gene targeting. Homologous intrachromosomal recombination between the repeated regions of the gene can reconstruct a functioning, wild-type gene. Treatment of these cells with the restriction endonuclease Xba I, which has a recognition site within the repeated region of HPRT homology, increased the frequency or homologous recombination bv more than 10-fold. Recombination frequency was similarly increased by treatment with the rare-cutting yeast endonuclease PI-Sce I when a cleavage site was placed within the repeated region of HPRT. In contrast, four restriction enzymes that cut at positions either outside of the repeated regions or between them produced no change in recombination frequency. The results suggest that homologous recombination between intrachromosomal repeats can be specifically initiated by a double-strand break occurring within regions of homology, consistent with the predictions of a model.     

Adeno-associated virus vector integration junctions

Vectors derived from adeno-associated virus (AAV) have the potential to stably transduce mammalian cells by integrating into host chromosomes. Despite active research on the use of AAV vectors for gene therapy, the structure of integrated vector proviruses has not previously been analyzed at the DNA sequence level. Studies on the integration of wild-type AAV have identified a common site-specific integration locus on human chromosome 19; however, most AAV vectors do not appear to integrate at this locus. To improve our understanding of AAV vector integration, we analyzed the DNA sequences of several integrated vector proviruses. HeLa cells were transduced with an AAV shuttle vector, and integrated proviruses containing flanking human DNA were recovered as bacterial plasmids for further analysis. We found that AAV vectors integrated as single-copy proviruses at random chromosomal locations and that the flanking HeLa DNA at integration sites was not homologous to AAV or the site-specific integration locus of wild-type AAV. Recombination junctions were scattered throughout the vector terminal repeats with no apparent site specificity. None of the integrated vectors were fully intact. Vector proviruses with nearly intact terminal repeats were excised and amplified after infection with wild-type AAV and adenovirus. Our results suggest that AAV vectors integrate by nonhomologous recombination after partial degradation of entering vector genomes. These findings have important implications for the mechanism of AAV vector integration and the use of these vectors in human gene therapy.     

Targeting of the Gi2 alpha gene in ES cells with replacement and insertion vectors

Five replacement vectors (RV) and one insertion vector (IV) were constructed in which ca. 10 kb of genomic Gi2 alpha sequence, flanked on one (IV) or both (RV) sides by a thymidine kinase (TK) marker, were disrupted by a Neo marker inserted into the NcoI site of exon 3. G418RFIAUR clones corresponding to ca. 4 x 10(8) ES cells electroporated with replacement vectors were analyzed and revealed no targeting event. The insertion vector, however, was integrated by a single reciprocal recombination resulting in a duplication of homology (Hit step; G418RFIAUS), which was lost--together with the plasmid and the TK sequences--by intrachromosomal recombination (Run step; G418RFIAUR). Thus, the Hit and Run strategy can be used with a selectable marker disrupting the targeted gene, giving rise to the same targeted product that would have been expected to arise from a double crossover with a replacement vector.     

Investigation of mycobacterial recA function: protein introns in the RecA of pathogenic mycobacteria do not affect competency for homologous recombination

The recA locus of pathogenic mycobacteria differs from that of non-pathogenic species in that it contains large intervening sequences termed protein introns or inteins that are excised by an unusual protein-splicing reaction. In addition, a high degree of illegitimate recombination has been observed in the pathogenic Mycobacterium tuberculosis complex. Homologous recombination is the main mechanism of integration of exogenous nucleic acids in M. smegmatis, a non-pathogenic mycobacterium species that carries an inteinless RecA and is amenable to genetic manipulations. To investigate the function of recA in mycobacteria, recA- strains of M. smegmatis were generated by allelic exchange techniques. These strains are characterized (i) by increased sensitivity towards DNA-damaging agents [ethylmethylsulphonate (EMS), mitomycin C, UV irradiation] and (ii) by the inability to integrate nucleic acids by homologous recombination. Transformation efficiencies using integrative or replicative vectors were not affected in recA- mutants, indicating that in mycobacteria RecA does not affect plasmid uptake or replication. Complementation of the recA- mutants with the recA from M. tuberculosis restored resistance towards EMS, mitomycin C and UV irradiation. Transformation of the complemented strains with suicide vectors targeting the pyrF gene resulted in numerous allelic exchange mutants. From these data, we conclude that the intein apparently does not interfere with RecA function, i.e. with respect to competency for homologous recombination, the RecAs from pathogenic and non-pathogenic mycobacteria are indistinguishable.     

Targeted recombination with single-stranded DNA vectors in mammalian cells

We studied the ability of single-stranded DNA (ssDNA) to participate in targeted recombination in mammalian cells. A 5' end-deleted adenine phosphoribosyltransferase (aprt) gene was subcloned into M13 vector, and the resulting ssDNA and its double-stranded DNA (dsDNA) were transfected to APRT-Chinese hamster ovary cells with a deleted aprt gene. APRT+ recombinants with the ssDNA was obtained at a frequency of 3 x 10(-7) per survivor, which was almost equal to that with the double-stranded equivalent. Analysis of the genome in recombinant clones produced by ssDNA revealed that 12 of 14 clones resulted from correction of the deletion in the aprt locus. On the other hand, the locus of the remaining 2 was not corrected; instead, the 5' deletion of the vector was corrected by end extension, followed by integration into random sites of the genome. To exclude the possibility that input ssDNA was converted into its duplex form before participating in a recombination reaction, we compared the frequency of extrachromosomal recombination between noncomplementary ssDNAs, and between one ssDNA and one dsDNA, of two phage vectors. The frequency with the ssDNAs was 0.4 x 10(-5), being 10-fold lower than that observed with the ssDNA and the dsDNA, suggesting that as little as 10% of the transfected ssDNA was converted into duplex forms before the recombination event, hence 90% remained unchanged as single-stranded molecules. Nevertheless, the above finding that ssDNA was as efficient as dsDNA in targeted recombination suggests that ssDNA itself is able to participate directly in targeted recombination reactions in mammalian cells.     

Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination

In mammalian cells, chromosomal double-strand breaks are efficiently repaired, yet little is known about the relative contributions of homologous recombination and illegitimate recombination in the repair process. In this study, we used a loss-of-function assay to assess the repair of double-strand breaks by homologous and illegitimate recombination. We have used a hamster cell line engineered by gene targeting to contain a tandem duplication of the native adenine phosphoribosyltransferase (APRT) gene with an I-SceI recognition site in the otherwise wild-type APRT+ copy of the gene. Site-specific double-strand breaks were induced by intracellular expression of I-SceI, a rare-cutting endonuclease from the yeast Saccharomyces cerevisiae. I-SceI cleavage stimulated homologous recombination about 100-fold; however, illegitimate recombination was stimulated more than 1,000-fold. These results suggest that illegitimate recombination is an important competing pathway with homologous recombination for chromosomal double-strand break repair in mammalian cells.     

Efficient modification of the APRT gene by FLP/FRT site-specific targeting

The FLP/FRT site-specific recombination system was established and characterized at the APRT gene in CHO cells. Targeting frequencies with FLP-stimulation were about 1 to 5 X 10(-5), which were 6-22-fold above gene targeting frequencies in the absence of FLP. Fifty two APRT+ cell lines were analyzed by Southern blotting: 56% were FLP-targeted integrants; 33% were APRT target convertants; 11% gave undefined patterns. In separate experiments we first enriched for integrants by screening for two additional markers carried on the targeting vector; 18 of 19 (95%) of the resulting cell lines were integrants. Intrachromosomal site-specific recombination was tested by reexposing integrants to FLP. Intrachromosomal popouts were stimulated over 200-fold, while homologous recombination in an adjacent interval was unchanged. The utility of this system was demonstrated by one-step FLP targeting to generate chromosomal substrates for homologous recombination, and by a two-step, FLP-and-run procedure to construct a chromosomal substrate for illegitimate recombination.     

I-SceI-induced gene replacement at a natural locus in embryonic stem cells

Gene targeting is a very powerful tool for studying mammalian development and physiology and for creating models of human diseases. In many instances, however, it is desirable to study different modifications of a target gene, but this is limited by the generally low frequency of homologous recombination in mammalian cells. We have developed a novel gene-targeting strategy in mouse embryonic stem cells that is based on the induction of endogenous gap repair processes at a defined location within the genome by induction of a double-strand break (DSB) in the gene to be mutated. This strategy was used to knock in an NH2-ezrin mutant in the villin gene, which encodes an actin-binding protein expressed in the brush border of the intestine and the kidney. To induce the DSB, an I-SceI yeast meganuclease restriction site was first introduced by gene targeting to the villin gene, followed by transient expression of I-SceI. The repair of the ensuing DSB was achieved with high efficiency (6 x 10[-6]) by a repair shuttle vector sharing only a 2.8-kb region of homology with the villin gene and no negative selection marker. Compared to conventional gene-targeting experiments at the villin locus, this represents a 100-fold stimulation of gene-targeting frequency, notwithstanding a much lower length of homology. This strategy will be very helpful in facilitating the targeted introduction of several types of mutations within a gene of interest.     

Gene knock-outs and allelic replacements in Toxoplasma gondii: HXGPRT as a selectable marker for hit-and-run mutagenesis

The hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) gene of the protozoan parasite Toxoplasma gondii encodes a safe, practical genetic marker suitable for both positive and negative selection. Taking advantage of the ability to control homologous versus nonhomologous recombination in haploid T. gondii tachyzoites by manipulating the length of homologous DNA sequence, we have explored the possibility of 'hit-and-run' mutagenesis to introduce gene knock-outs (or allelic replacements) at loci for which no known selection or screen is available. Using the uracil phosphoribosyl transferase (UPRT) locus as a target, a genomic clone containing approximately 8 kb encompassing the UPRT gene (but lacking essential coding sequence) was fused to a cDNA-derived HXGPRT 'minigene', which lacks sufficient contiguous genomic sequence for homologous recombination. After transfection of circular plasmid DNA, positive selection for HXGPRT activity identified stable transformants, > 30% of which were found to have integrated at the UPRT locus as 'pseudodiploids' (produced by single-site homologous recombination between the circular plasmid and genomic DNA). Upon removal of mycophenolic acid, resolution of pseudodiploids by spontaneous intrachromosomal homologous recombination was selected using 6-thioxanthine, yielding a 1:1 ratio of UPRT knock-out parasites to wild-type revertants, at frequencies of approximately 10(-6) per parasite doubling. Applications of 'hit-and-run' technology relative to other gene targeting strategies are discussed.     

Titrating methylphenidate in children with attention-deficit/hyperactivity disorder: is body mass predictive of clinical response?

We report here on strategies aimed at improving the frequency of detectable recombination in plants by increasing the efficiency of selecting double-recombinants in transgenic calli. Gene targeting was approached on the Gln1 and the Pzfloci of Lotus japonicus, using Agrobacterium tumefaciens T-DNA replacement vectors. Large flanking regions, up to 22.9 kb, surrounding a positive selection marker were presented as substrates for homologous recombination. For easier detection of putative recombinants the negative selectable marker cytosine deaminase was inserted at the outside borders of the flanking regions offered for cross-over. A combination of positive and negative selection allowing double-recombinants to grow, while counter-selecting random insertions, was used to select putative targeting events. The more than 1000-fold enrichment observed with replacement vectors designed to minimize gene silencing demonstrated the efficiency of the negative selection. Using five different replacement vectors an estimated total of 18,974 transformation events were taken through the positive-negative selection procedure and 185 resistant calli obtained. Targeting events could not be verified in the survivors by PCR screening and Southern blot analysis. With this approach the frequency of detectable gene targeting in L. japonicus was below 5.3 x 10(-5), despite the large flanking sequences offered for recombination.     

Clinical value of ambulatory monitoring of blood pressure]

Stable transduction of mammalian cells typically involves random integration of viral vectors by non-homologous recombination. Here we report that vectors based on adeno-associated virus (AAV) can efficiently modify homologous human chromosomal target sequences. Both integrated neomycin phosphotransferase genes and the hypoxanthine phosphoribosyltransferase gene were targeted by AAV vectors. Site-specific genetic modifications could be introduced into approximately 1% of cells, with the highest targeting rates occurring in normal human fibroblasts. These results suggest that AAV vectors could be used to introduce specific genetic changes into the genomic DNA of a wide variety of mammalian cells, including therapeutic gene targeting applications.     

Gene targeting in Penicillium chrysogenum: disruption of the lys2 gene leads to penicillin overproduction

Two strategies have been used for targeted integration at the lys2 locus of Penicillium chrysogenum. In the first strategy the disruption of lys2 was obtained by a single crossing over between the endogenous lys2 and a fragment of the same gene located in an integrative plasmid. lys2-disrupted mutants were obtained with 1.6% efficiency when the lys2 homologous region was 4.9 kb, but no homologous integration was observed with constructions containing a shorter homologous region. Similarly, lys2-disrupted mutants were obtained by a double crossing over (gene replacement) with an efficiency of 0.14% by using two lys2 homologous regions of 4.3 and 3.0 kb flanking the pyrG marker. No homologous recombination was observed when the selectable marker was flanked by short lys2 homologous DNA fragments. The disruption of lys2 was confirmed by Southern blot analysis of three different lysine auxotrophs obtained by a single crossing over or gene replacement. The lys2-disrupted mutants lacked alpha-aminoadipate reductase activity (encoded by lys2) and showed specific penicillin yields double those of the parental nondisrupted strain, Wis 54-1255. The alpha-aminoadipic acid precursor is channelled to penicillin biosynthesis by blocking the lysine biosynthesis branch at the alpha-aminoadipate reductase level.     

Two distinct apolipoprotein B alleles in mice generated by a single 'in-out' targeting

'In-out' gene targeting using a hypoxanthine phosphoribosyltransferase (HPRT) minigene was applied to generate two new alleles in the gene (Apob) coding for apolipoprotein B (apo B) in murine embryonic stem (ES) cells. Homologous integration of the targeting vector during the 'in step' disrupted the Apob gene leading to an allele encoding apo B81, having a 19% carboxyl-terminal truncation. All six targeted cells obtained had more than one insert at the locus, and the chromosomal target sequence in four of them was changed during the recombination. These results suggest that concatenation of the targeting vector prior to insertion was needed to generate sufficient gene product to yield the HPRT+ phenotype, and that recombination between the concatenated DNA and endogenous DNA was a gene replacement more frequently than a simple insertion. The 'out step' recombination event which occurs between sequences duplicated in the 'in step', was planned to replace the sequences encoding the putative LDL receptor-binding domains of apo B100 with sequences encoding human beta-globin peptides (designated apo B100-beta). 6-Thioguanine (6-TG) resistant colonies were obtained from all the 'in-step' cell lines tested at frequencies of 10(-5) to 10(-4), but the frequency of physical loss of the HPRT sequences accompanied by retention of the modified Apob sequence was variable, indicating that mechanisms other than a simple excision are responsible for the generation of 6-TG resistance. Mice from the 'in-step' produce apo B81 and display characteristics of familial hypobetalipoproteinemia; some homozygotes develop hydrocephaly or exencephaly. Mice from the 'out-step' produce apo B100-beta and secrete lipoprotein particles containing the modified protein; their phenotypic changes are subtle, suggesting the lack of the putative LDL receptor-binding domains is not sufficient to increase the steady-state level of apo B100-beta particles above that of apo B100 particles in control mice.