Biomedical subjects

P Hasty

Publications and source records attributed to P Hasty.

At least 19 recordsLinked to original sources

PubMed1999

Temporal, spatial and tissue-specific expression of a myogenin-lacZ transgene targeted to the Hprt locus in mice.

A lacZ transgene, expressed by the myogenin promoter, was introduced into the mouse hypoxanthine phosphoribosyltransferase (Hprt) locus by gene targeting in embryonic stem cells. Embryos between E10.5-E18.5 days were analyzed for expression of the transgene after staining for beta-galactosidase activity. Transgene expression was restricted to the skeletal muscle lineages reflecting a similar temporal and spatial pattern previously demonstrated for the endogenous myogenin gene. Additionally, a second transgene, MC1tk, showed expression in 87% of the clones when targeted to Hprt. This strategy, called targeted transgenesis, provides control for analyzing promoter sequences and for comparing various transgenes expressed by the same promoter.

Animals↗

PubMed1998

Cells deleted for Brca2 COOH terminus exhibit hypersensitivity to gamma-radiation and premature senescence.

The putative Brca2-MmRad51 interaction is analyzed in mouse cells deleted for the COOH terminus of Brca2 (amino acids 3140-3328), which contains a region that associates with MmRad51 by yeast two-hybrid. These cells are hypersensitive to gamma-radiation (suggesting defective recombinational repair) but not UV light (suggesting intact nucleotide excision repair) and maintain the G1-S and G2-M checkpoints after exposure to gamma-irradiation. Cells deleted for the COOH terminus of Brca2 progress through the cell cycle at a similar rate as wild-type cells but undergo senescence more rapidly. These data support the hypothesis that deletion of Brca2 stimulates cancer by defective MmRad51-mediated DNA repair and not by defective cell cycle regulation.

Animals↗

PubMed1997

Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking Brca2.

Inherited mutations in the human BRCA2 gene cause about half of the cases of early-onset breast cancer. The embryonic expression pattern of the mouse Brca2 gene is now defined and an interaction identified of the Brca2 protein with the DNA-repair protein Rad51. Developmental arrest in Brca2-deficient embryos, their radiation sensitivity, and the association of Brca2 with Rad51 indicate that Brca2 may be an essential cofactor in the Rad51-dependent DNA repair of double-strand breaks, thereby explaining the tumour-suppressor function of Brca2.

3T3 Cells↗

PubMed1997

Targeted mutation in beta1,4-galactosyltransferase leads to pituitary insufficiency and neonatal lethality.

Despite much attention, the function of oligosaccharide chains on glycoproteins and glycolipids remains largely unknown. Our understanding of oligosaccharide function in vivo has been limited to the use of reagents and targeted mutations that eliminate entire classes of oligosaccharide chains. However, most biological functions for oligosaccharides have been attributed to specific terminal sequences on these glycoside chains; yet, there have been few studies that examine the consequences of modifying terminal oligosaccharide structures in vivo. To address this issue, mice were created bearing a targeted mutation in beta1,4-galactosyltransferase (GalTase), an enzyme responsible for elaboration of many of the proposed biologically active carbohydrate epitopes. Most GalTase-null mice died within the first few weeks after birth and were characterized by stunted growth, thin skin, sparse hair, and dehydration. In addition, spermatogenesis was delayed, the lungs were poorly developed, and the adrenal cortices were poorly stratified. The few surviving adults had puffy skin (myxedema) and difficulty delivering pups at birth (dystocia) and failed to lactate (agalactosis). All of these defects are consistent with endocrine insufficiency, which was confirmed by markedly decreased levels of serum thyroxine. The polyglandular nature of the endocrine insufficiency is indicative of a failure of the anterior pituitary gland to stimulate the target endocrine organs. Previous in vitro studies have suggested that incomplete glycosylation of anterior pituitary hormones leads to the creation of hormone antagonists, which down-regulate subsequent endocrine function, producing polyglandular endocrine insufficiency. In GalTase-null mice, the anterior pituitary acquired a normal secretory phenotype during neonatal development indicative of normal glycoprotein hormone synthesis and secretion. However, as expected, the gland was devoid of GalTase activity. These results support a requirement for terminal oligosaccharide sequences for anterior pituitary hormone function. The fact that approximately 10% of the GalTase-null mice survive the neonatal period indicates the presence of a previously unrecognized compensatory pathway for glycoprotein hormone glycosylation and/or action.

Abnormalities, Multiple↗

PubMed1996

Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates.

Ku is a heterodimeric DNA end binding complex composed of 70 and 86 kDa subunits. Here, we show that Ku86 is essential for normal V(D)J recombination in vivo, as Ku86-deficient mice are severely defective for formation of coding joints. Unlike severe combined immunodeficient (scid) mice, Ku86-deficient mice are also defective for signal joint formation. Both hairpin coding ends and blunt full-length signal ends accumulate. Contrary to expectation, Ku86 is evidently not required for protection of either type of V(D)J recombination intermediate. Instead, V(D)J recombination appears to be arrested after the cleavage step in Ku86-deficient mice. We suggest that Ku86 may be required to remodel or disassemble DNA-protein complexes containing broken ends, making them available for further processing and joining.

Animals↗

PubMed1996

A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53.

RecA in Escherichia coli and its homolog, ScRad51 in Saccharomyces cerevisiae, are known to be essential for recombinational repair. The homolog of RecA and ScRad51 in mice, MmRad51, was mutated to determine its function. Mutant embryos arrested early during development. A decrease in cell proliferation, followed by programmed cell death and chromosome loss, was observed. Radiation sensitivity was demonstrated in trophectoderm-derived cells. Interestingly, embryonic development progressed further in a p53 null background; however, fibroblasts derived from double-mutant embryos failed to proliferate in tissue culture.

Animals↗

PubMed1995

Gene conversion during vector insertion in embryonic stem cells.

Recombination of an insertion vector into its chromosomal homologue is a conservative event in that both the chromosomal and the vector sequences are preserved. However, gene conversion may accompany homologous recombination of an insertion vector. To examine gene conversion in more detail we have determined the targeting frequencies and the structure of the recombinant alleles generated with a series of vectors which target the hprt gene in embryonic stem cells. We demonstrate that gene conversion of the introduced mutation does not significantly limit homologous recombination and that gene conversion occurs without a sequence specific bias for five different mutations. The frequency of the loss of a vector mutation and the gain of a chromosomal sequence is inversely proportional to the distance between the vector mutation and the double-strand break. The loss of a chromosomal sequence and the gain of a vector mutation occurs at a low frequency.

Alleles↗

PubMed1995

Severe phenotype in mice with termination mutation in exon 2 of cystic fibrosis gene.

Mice with a termination codon mutation in exon 2 of the cystic fibrosis (CF) gene were generated using homologous recombination in embryonic stem cells. Animals homozygous for the mutant allele display a severe intestinal phenotype similar to that previously reported for CF mutant mice. The null nature of this allele was demonstrated by the absence of detectable wild-type mRNA, by the absence of detectable CFTR in the serous gland collecting ducts of salivary tissues, and by the lack of cAMP-mediated short-circuit current responses in colonic epithelium of mutant animals.

Animals↗

PubMed1995

Gene targeting in mouse embryonic stem cells with an adenoviral vector.

We examined the ability of an E1, E3-defective adenoviral vector to act as a substrate for homologous recombination with chromosomal DNA by including host chromosomal sequence from the mouse Fgr locus that also contained a selectable marker. After infection of mouse embryonic stem cells, stable integration was selected for neomycin resistance and the efficiency of homologous recombination was evaluated. The adenoviral vector was capable of infecting mouse embryonic stem cells efficiently. Between 30-50% of the input virus reached the nuclei after 24 hours of infection. Surprisingly, even without negative selection, 25-40% of the integration resulted from homologous recombination at m.o.i. 10 and 100, although the absolute efficiency of integration was low. Our results suggest that it is possible to modify the structure of an adenoviral vector to achieve a high gene targeting efficiency, resulting in regulated and long-term expression of an introduced gene.

Adenoviridae↗

PubMed1994

Efficiency of insertion versus replacement vector targeting varies at different chromosomal loci.

We have analyzed the targeting frequencies and recombination products generated with isogenic vectors at the fah and fgr loci in embryonic stem cells. A single vector which could be linearized at different sites to generate either a replacement or an insertion vector was constructed for each locus. A replacement event predominated when the vectors were linearized at the edge of the homologous sequences, while an insertion event predominated when the vectors were linearized within the homologous sequences. However, the ratio of the targeting frequencies exhibited by the different vector configurations differed for the two loci. When the fgr vector was linearized as an insertion vector, the ratio of targeted to random integrations was four- to eightfold greater than when the vector was linearized as a replacement vector. By contrast, the ratio of targeted to random integrations at the fah locus did not vary with the linearization site of the vector. The different relationships between the targeting frequency and the vector configuration at the fgr and fah loci may indicate a DNA sequence or chromatin structure preference for different targeting pathways.

Animals↗

PubMed1994

Targeting frequency for deletion vectors in embryonic stem cells.

We analyzed the gene targeting frequencies and recombination products generated by a series of replacement deletion vectors which target the hprt (hypoxanthine phosphoribosyltransferase) locus in mouse embryonic stem cells. We found that the targeting frequency of a 19.2-kb deletion was comparable to that of a 3-kb deletion or a conventional replacement event in which a 1.7-kb fragment was inserted into the locus. We also observed different integration patterns for these deletion vectors. A result of this finding is that a wide range of genomic deletions in embryonic stem cells is feasible.

Animals↗

PubMed1993

Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene.

Myogenin is a muscle-specific transcription factor that can induce myogenesis in a variety of cell types in tissue culture. To test myogenin's role in vivo, mice homozygous for a targeted mutation in the myogenin gene were generated. These mice survive fetal development but die immediately after birth and show a severe reduction of all skeletal muscle. Myogenin-mutant mice differ from mice carrying mutations in genes for the related myogenic factors Myf5 and MyoD, which have no muscle defects. Myogenin is therefore essential for the development of functional skeletal muscle.

Animals↗

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↗