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Biomedical subjects

M R Capecchi

Publications and source records attributed to M R Capecchi.

At least 73 records · Page 4Linked to original sources
PubMed1992

Developmental defects of the ear, cranial nerves and hindbrain resulting from targeted disruption of the mouse homeobox gene Hox-1.6.

Gene targeting in mouse embryo-derived stem cells has been used to generate mice with a disruption in the homeobox gene Hox-1.6. Mice heterozygous at the Hox-1.6 locus appear normal, whereas Hox-1.6-/Hox-1.6- mice die at or shortly after birth. These homozygotes exhibit profound defects in the formation of the external, middle and inner ears as well as in specific hindbrain nuclei, and in cranial nerves and ganglia. The affected tissues lie within a narrow region along the anteroposterior axis of the mouse but are of diverse embryonic origin. The set of defects associated with the disruption of Hox-1.6 is distinct from and nonoverlapping with that of the closely linked Hox-1.5 gene. But both mutations cause loss, rather than homeotic transformation, of tissues and structures.

Animals↗
PubMed1992

High-fidelity gene targeting in embryonic stem cells by using sequence replacement vectors.

Mutations were targeted to the Hprt locus in murine embryonic stem cells by using sequence replacement vectors. When the vector was designed such that the mutated sequences were flanked on both sides by several kilobases of DNA homologous to the target locus, replacement of chromosomal sequences with the exogenous DNA occurred with precision. If, on the other hand, the target-homologous DNA on one arm of the vector was reduced to below 1 kb in length, the fidelity of recombination was diminished.

Animals↗
PubMed1992

Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus.

Mutations were targeted to the Hprt locus of mouse embryo-derived stem cells by using 22 different sequence replacement and sequence insertion vectors. The targeting frequency was examined at two sites within the Hprt locus as a function of the extent of homology between the targeting vector and the target locus. The targeting frequency was also compared by using vectors prepared from isogenic and nonisogenic DNA sources. With one exception, all of the vectors showed the same exponential dependence of targeting efficiency on the extent of homology between the targeting vector and the target locus. This was true regardless of whether they were sequence replacement or sequence insertion vectors, whether they were directed toward either of the two different sites within the Hprt locus, or whether they were prepared from isogenic or nonisogenic DNA sources. Vectors prepared from isogenic DNA targeted four to five times more efficiently than did the corresponding vectors prepared from nonisogenic DNA. The single case of unexpectedly low targeting efficiency involved one of the vectors prepared from nonisogenic DNA and could be attributed to an unfavorable distribution of heterology between the Hprt sequences present in the targeting vector and the endogenous Hprt gene.

Animals↗
PubMed1991

Swaying is a mutant allele of the proto-oncogene Wnt-1.

Mice homozygous for the recessive mutation swaying (sw) are characterized by ataxia and hypertonia, attributed to the malformation of anterior regions of the cerebellum. We show that sw is a deletion of a single base pair from the proto-oncogene Wnt-1. The deletion is predicted to cause premature termination of translation, eliminating the carboxy-terminal half of the Wnt-1 protein. Histological examination shows that sw is phenotypically identical to a previously described wnt-1 mutation introduced into mice by gene targeting. Although both mutations in Wnt-1 disrupt primarily the development of the anterior cerebellum, they also exhibit a variability in expressivity such that rostrally adjacent structures in the midbrain and caudally adjacent structures in the posterior cerebellum can also be affected.

Alleles↗
PubMed1991

Regionally restricted developmental defects resulting from targeted disruption of the mouse homeobox gene hox-1.5.

Gene targeting in mouse embryo-derived stem cells has been used to disrupt the homeobox gene hox-1.5. Mice heterozygous at the hox-1.5 locus appear normal, whereas hox-1.5-/hox-1.5- mice die at or shortly after birth. These homozygotes are athymic, aparathyroid, have reduced thyroid and submaxillary tissue and exhibit a wide range of throat abnormalities. In addition, they often feature defects of the heart and arteries as well as craniofacial abnormalities. These deficiencies are remarkably similar to the pathology of the human congenital disorder DiGeorge's syndrome.

Abnormalities, Multiple↗
PubMed1990

Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development.

The int-1 proto-oncogene was first identified as a gene activated in virally induced mouse mammary tumours. Expression studies, however, suggest that the normal function of this gene may be in spermatogenesis and in the development of the central nervous system. Genes sharing sequence similarity with int-1 have been found throughout the animal kingdom. For example, int-1 has 54% amino-acid identity to the Drosophila segment polarity gene wingless (wg). Both the int-1 and wg gene products seem to be secreted proteins, presumably involved in cell-cell signalling. We have now explored the function of int-1 in the mouse by disrupting one of the two int-1 alleles in mouse embryo-derived stem cells using positive-negative selection. This cell line was used to generate a chimaeric mouse that transmitted the mutant allele to its progeny. Mice heterozygous for the int-1 null mutation are normal and fertile, whereas mice homozygous for the mutation may exhibit a range of phenotypes from death before birth to survival with severe ataxia. The latter pathology in mice and humans is often associated with defects in the cerebellum. Examination of int-1-/int-1- mice at several stages of embryogenesis revealed severe abnormalities in the development of the mesencephalon and metencephalon indicating a prominent role for the int-1 protein is in the induction of the mesencephalon and cerebellum.

Animals↗
PubMed1990

Introduction of a lacZ reporter gene into the mouse int-2 locus by homologous recombination.

We demonstrate that the frequency of gene targeting is unaffected by the length of nonhomologous DNA transferred to a target chromosomal sequence. A result of this finding is that a much wider spectrum of designed genomic alterations is now feasible. As a first application, we inserted a 5.4-kilobase cassette of nonhomologous DNA into the int-2 locus in mouse embryo-derived stem cells by gene targeting. The inserted DNA contained a lacZ gene positioned to create an in-frame fusion with the int-2 protein-coding region. Upon differentiation of these cells to embryoid bodies, the int-2-lacZ fusion faithfully reproduced the expression pattern of int-2 RNA. This ability to target reporter genes, such as lacZ, to specific mouse loci, combined with the ability to move the tagged gene into different mutant backgrounds, may provide an ideal approach for analyzing interactions among genes that participate in a developmental network.

Animals↗
PubMed1989

Altering the genome by homologous recombination.

Homologous recombination between DNA sequences residing in the chromosome and newly introduced, cloned DNA sequences (gene targeting) allows the transfer of any modification of the cloned gene into the genome of a living cell. This article discusses the current status of gene targeting with particular emphasis on germ line modification of the mouse genome, and describes the different methods so far employed to identify those rare embryonic stem cells in which the desired targeting event has occurred.

Animals↗
PubMed1989

The new mouse genetics: altering the genome by gene targeting.

Gene targeting (homologous recombination between DNA sequences residing in the chromosome and newly introduced DNA sequences) in pluripotent, mouse embryo-derived stem (ES) cells promises to provide the means to generate mice of any desired genotype. This review describes some of the background and current advances of gene targeting in mouse ES cells.

Animals↗
PubMed1988

Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes.

Gene targeting--homologous recombination of DNA sequences residing in the chromosome with newly introduced DNA sequences--in mouse embryo-derived stem cells promises to provide a means to generate mice of any desired genotype. We describe a positive nd negative selection procedure that enriches 2,000-fold for those cells that contain a targeted mutation. The procedure was applied to the isolation of hprt- and int-2- mutants, but it should be applicable to any gene.

Animals↗
PubMed1987

Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells.

We mutated, by gene targeting, the endogenous hypoxanthine phosphoribosyl transferase (HPRT) gene in mouse embryo-derived stem (ES) cells. A specialized construct of the neomycin resistance (neor) gene was introduced into an exon of a cloned fragment of the Hprt gene and used to transfect ES cells. Among the G418r colonies, 1/1000 were also resistant to the base analog 6-thioguanine (6-TG). The G418r, 6-TGr cells were all shown to be Hprt- as the result of homologous recombination with the exogenous, neor-containing, Hprt sequences. We have compared the gene-targeting efficiencies of two classes of neor-Hprt recombinant vectors: those that replace the endogenous sequence with the exogenous sequence and those that insert the exogenous sequence into the endogenous sequence. The targeting efficiencies of both classes of vectors are strongly dependent upon the extent of homology between exogenous and endogenous sequences. The protocol described herein should be useful for targeting mutations into any gene.

Animals↗
PubMed1986

High frequency targeting of genes to specific sites in the mammalian genome.

We corrected a defective gene residing in the chromosome of a mammalian cell by injecting into the nucleus copies of the same gene carrying a different mutation. We determined how the number, the arrangement, and the chromosomal position of the integrated gene, as well as the number of injected molecules influence the gene-targeting frequency. Recombination between the newly introduced DNA and its chromosomal homolog occurred at a frequency of 1 in 10(3) cells receiving DNA. Correction events were mediated by either double reciprocal recombination or gene conversion. This resulted in sequences in the genome being replaced by sequences of the introduced DNA or, in separate experiments, sequences in the incoming DNA being replaced by chromosomal sequences. Both point mutations and deletion mutations were corrected; however, the nature of the mutation carried by the respective sequence influenced whether the integrated or injected sequence was corrected.

Alleles↗
PubMed1986

Analysis of homologous recombination in cultured mammalian cells in transient expression and stable transformation assays.

Recombination between plasmid molecules, each containing a nonoverlapping deletion mutation in the hamster adenine phosphoribosyltransferase gene, was measured after coinjection into rat cells. Using these two plasmids, as linear or circular molecules, the recombination efficiency was measured soon after injection in a transient expression assay or after selection for stable transformants. The transient assay revealed that linear molecules were a better substrate for recombination, with double strand breaks within the region of homology stimulating recombination more than breaks outside the region of homology. A 20 to 70-fold increase in the efficiency of recombination was observed when two linear molecules were coinjected as compared to two circular molecules. Linear molecules were found to not only stimulate recombination but also to facilitate stable integration of the recombinant molecule into the host genome.

Animals↗
PubMed1986

Isolation and characterization of Caenorhabditis elegans DNA sequences homologous to the v-abl oncogene.

DNA sequences homologous to the v-abl oncogene were isolated from a Caenorhabditis elegans genomic library by their ability to hybridize with a v-src probe. The DNA sequence of 2465 nucleotides of one clone was determined. This region corresponds to the 5' protein kinase domain of v-abl plus approximately equal to 375 base pairs toward the 3' end. Four potential introns were identified. The homology between the deduced amino acid sequence of the C. elegans clone and that of the 1.2-kilobase-pair protein kinase region of v-abl is 62%. The tyrosine residue corresponding to the tyrosine that is phosphorylated in the v-src protein is conserved in the C. elegans sequence. When 95 amino acids around this tyrosine were compared with the corresponding sequences of Drosophila c-abl, v-abl, and v-src, the identities were 83%, 79%, and 56%, respectively. Hybridization of the cloned DNA with C. elegans poly(A)+ RNA revealed a major transcript of 4.4 kilobases.

Amino Acid Sequence↗
PubMed1985

Expression of a microinjected porcine class I major histocompatibility complex gene in transgenic mice.

A porcine class I major histocompatibility complex (SLA) gene has been introduced into the genome of a C57BL/10 mouse. This transgenic mouse expressed SLA antigen on its cell surfaces and transmitted the gene to offspring, in which the gene is also expressed. Skin grafts of such transgenic mice were rejected by normal C57BL/10 mice, suggesting that the foreign SLA antigen expressed in the transgenic mice is recognized as a functional transplantation antigen.

Animals↗
PubMed1985

Effect of cell cycle position on transformation by microinjection.

We have investigated the effect of cell cycle position on the efficiency of transformation by microinjection. Linear recombinant plasmids transform synchronized cells with similar frequencies following injections at all cell cycle stages, whereas supercoiled molecules show a decreased ability to generate transformants in early S phase. This inhibition is not due to an inability to transiently express a transferred gene, since cells at all stages of the cycle efficiently expressed a hamster adenine phosphoribosyltransferase gene introduced on a supercoiled plasmid. Southern transfer analyses of the cell cycle specific transformants revealed that tandem arrays of plasmids, integrated into the host chromosomes, could be generated at all cell cycle stages.

Adenine Phosphoribosyltransferase↗