Biomedical subjects

P Hasty

Publications and source records attributed to P Hasty.

28 records · Page 2Linked to original sources

PubMed1993

Disruption of the G(i2) alpha locus in embryonic stem cells and mice: a modified hit and run strategy with detection by a PCR dependent on gap repair.

We have used an insertion vector-based approach to target the G(i2) alpha gene in AB-1 embryonic stem cells. 105 bp located 0.8-0.9 kb upstream of a disrupting Neo marker in exon 3 were deleted and replaced with an engineered Not I site, that served to linearize the vector. The 105 bp deletion served as a primer annealing site in a polymerase chain reaction (PCR) designed to detect the gap repair associated with homologous recombination. Both target conversion and vector insertion events were obtained ('hit' step). Clones that had inserted the entire targeting vector were taken into FIAU (1-[2-deoxy,2-fluoro-beta-D-arabinofuranosyl]-5-ioduracil) counterselection to select against a thymidine kinase (TK) marker flanking the homologous genomic sequences and thus for cells that had excised the plasmid and the TK marker by intrachromosomal recombination ('run' step). Additional selection in G418 reduced the number of drug-resistant colonies at least five-fold. Thus, the Neo marker disrupting the homologous sequences allows for a more specific selection of the desired intrachromosomal recombination event in tissue culture. This modified 'hit and run' strategy represents a novel approach for vector design and the use of the polymerase chain reaction to detect targeting. It may be particularly useful for targeting genes that display a low frequency of homologous recombination. Germ line transmission of the mutated G(i2) alpha allele is also demonstrated.

Animals↗

PubMed1993

A severe phenotype in mice with a duplication of exon 3 in the cystic fibrosis locus.

To develop an animal model for cystic fibrosis (CF), targeted gene disruption in embryonic stem (ES) cells was used to generate a duplication of exon 3 (cftrm1Bay allele) of the mouse CF gene. ES cells containing this mutation were used to generate chimeric animals that transmitted the mutant allele through the germline. Homozygous mutant animals display a severe phenotype, with approximately 40% dying within 1 week from intestinal obstruction. RNAase protection analysis of the cftrm1Bay allele did not detect any normal mRNA (< 1-2% of wild-type) in mutant animals. Pathologic changes in the intestines from mutant mice included mucus accumulation in the crypts and intestinal lumen, dilatation of the bases of the crypts, enlargement of goblet cells, and the presence of concretions in the crypts or between the villi. Changes were also present in the mucosal glands of the pharynx and the minor sublingual glands, where dilatation of acini and accumulation of eosinophilic material were evident. Atrophy of acinar cells that may be secondary to nutritional deficiency and mild inflammation in the main pancreatic duct were present in the pancreas of mutant animals. No changes were noted in the lung, trachea, liver, or male reproductive tract of mutant animals, and mutant males were fertile. Homozygous mutant mice showed defects in cAMP-mediated ion transport both in ileum and in cultured fetal tracheal explants. Thus, an additional mouse model for CF has been generated that should prove useful for the understanding of the pathogenesis and the development of treatments for CF.

Alleles↗

PubMed1993

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.

Animals↗

PubMed1992

Genetic manipulation of the mouse via gene targeting in embryonic stem cells.

Gene targeting applied to totipotent embryonic stem (ES) cells is a very powerful means of creating highly specific mutations of genes in the mouse. The successful application of this technology is however constrained by both the types of mutations that can be generated at a target locus and the ability to reconstruct a germline chimera from the manipulated cells. We have developed two cell lines that can be routinely transmitted through the germline of chimeras after cloning and prolonged selection in tissue culture. We have also established a variety of methods for generating non-selected mutations at the X-linked hprt locus in ES cells. Our observations at this locus have enabled us to generate successfully a subtle mutation at the non-selectable Hox-2.6 locus.

Animals↗

PubMed1992

Modifying the mouse: design and desire.

Genetic modification of endogenous genes in mice has become possible by applying gene targeting techniques to embryonic stem (ES) cells and using specific clones of cells to generate mice. Despite the experimental opportunities offered by the creation of organisms with specific genetic changes, there are considerable technical obstacles which can confound the routine implementation of this technology. This review addresses some recent advances in the ability to construct mice with a variety of genetic modifications. These include an increased understanding of the basic cell biology and in vitro growth characteristics of ES cells, which has facilitated germ line transmission of manipulated clones on a routine basis. The techniques that are used to isolate "targeted" clones of ES cells have been summarized, and the current status of strategies which have been successfully used to make very specific modifications of the genome are discussed.

Alleles↗

PubMed1992

The role and fate of DNA ends for homologous recombination in embryonic stem cells.

We have analyzed the gene-targeting frequencies and recombination products generated by a series of vectors which target the hprt locus in embryonic stem cells and found the existence of alternative pathways that depend on the location of the double-strand break within the vector. A double-strand break in the targeting homology was found to increase the targeting frequency compared with a double-strand break at the edge of or outside the target homology; this finding agrees with the double-strand break repair model proposed for Saccharomyces cerevisiae. Although a double-strand break in the homology is important for efficient targeting, observations reported here suggest that the terminal ends are not always directly involved in the initial recombination event. Short terminal heterologous sequences which block the homologous ends of the vector may be incorporated into the target locus. A modification of the double-strand break repair model is described to account for this observation.

Animals↗

PubMed1991

Fidelity of targeted recombination in human fibroblasts and murine embryonic stem cells.

Targeted recombination in murine embryonic stem cells promises to be a powerful tool for introducing specific mutations into target genes to study development in mice and to create animal models of human disease. Gene targeting also holds potential for correcting genetic defects as an approach to human gene therapy. To precisely modify target genes, homologous recombination must proceed with high fidelity. However, several results have suggested that targeted recombination may be highly mutagenic. To test the accuracy of gene targeting we analyzed 44 independent targeted recombinants at the hypoxanthine phosphoribosyltransferase (HPRT) locus in a human fibroblast cell line and in mouse embryonic stem cells. We surveyed 80 kilobases around the sites of recombination by using chemical cleavage of mismatches. Only two mutations were found: a T----G transversion and a thymidine deletion. Thus, gene targeting in mammalian cells can be extremely accurate. These results demonstrate the feasibility of generating precise modifications of mammalian genomes by gene targeting.

Animals↗

PubMed1991

Introduction of a subtle mutation into the Hox-2.6 locus in embryonic stem cells.

Gene targeting in embryonic stem (ES) cells is a powerful tool for generating mice with null alleles. Current methods of gene inactivation in ES cells introduce a neomycin gene (neo) cassette both as a mutagen and a selection marker for transfected cells. Although null alleles are valuable, changes at the nucleotide level of a gene are very important for functional analysis. One gene family in which subtle mutations would be particularly valuable are the clusters of Hox homeobox genes. Inactivation of gene in a cluster with a neo cassette that includes promoter/enhancer elements may deregulate transcription of neighbouring genes and generate a phenotype which is difficult to interpret. We describe here a highly efficient gene targeting method, termed the 'hit and run' procedure. This generates ES cells with subtle site-specific mutations with no selectable marker and may be useful for most genes. We have developed this procedure at the hypoxanthine phosphoribosyltransferase (hprt) locus and subsequently isolated ES cells with a premature stop codon in the homeobox of Hox-2.6 (ref. 14).

Alleles↗

PubMed1991

The length of homology required for gene targeting in embryonic stem cells.

Homologous recombination has been used to introduce site-specific mutations into murine embryonic stem (ES) cells with both insertion and replacement vectors. In this study, we compared the frequency of gene targeting with various lengths of homology and found a dramatic increase in targeting with an increase in homology from 1.3 to 6.8 kb. We examined in detail the relationship between the length of homology and the gene-targeting frequency for replacement vectors and found that a critical length of homology is needed for targeting. Adding greater lengths of homology to this critical length has less of an effect on the targeting frequency. We also analyzed the lengths of homology necessary on both arms of the vector for gene replacement events and found that 472 bp of homology is used as efficiently as 1.2 kb in the formation and resolution of crossover junctions.

Animals↗

PubMed1991

Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells.

Gene targeting has been used to direct mutations into specific chromosomal loci in murine embryonic stem (ES) cells. The altered locus can be studied in vivo with chimeras and, if the mutated cells contribute to the germ line, in their offspring. Although homologous recombination is the basis for the widely used gene targeting techniques, to date, the mechanism of homologous recombination between a vector and the chromosomal target in mammalian cells is essentially unknown. Here we look at the nature of gene targeting in ES cells by comparing an insertion vector with replacement vectors that target hprt. We found that the insertion vector targeted up to ninefold more frequently than a replacement vector with the same length of homologous sequence. We also observed that the majority of clones targeted with replacement vectors did not recombine as predicted. Analysis of the recombinant structures showed that the external heterologous sequences were often incorporated into the target locus. This observation can be explained by either single reciprocal recombination (vector insertion) of a recircularized vector or double reciprocal recombination/gene conversion (gene replacement) of a vector concatemer. Thus, single reciprocal recombination of an insertion vector occurs 92-fold more frequently than double reciprocal recombination of a replacement vector with crossover junctions on both the long and short arms.

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