Biomedical subjects

Carl A Pinkert

Publications and source records attributed to Carl A Pinkert.

9 recordsLinked to original sources

PubMed2005

Microinjection of cytoplasm or mitochondria derived from somatic cells affects parthenogenetic development of murine oocytes.

Cloned mammals are readily obtained by nuclear transfer using cultured somatic cells; however, the rate of generating live offspring from the reconstructed embryos remains low. In nuclear transfer procedures, varying quantities of donor cell mitochondria are transferred with nuclei into recipient oocytes, and mitochondrial heteroplasmy has been observed. A mouse model was used to examine whether transferred mitochondria affect the development of the reconstructed oocytes. Cytoplasm or purified mitochondria from somatic cells derived from the external ear, skeletal muscle, and testis of Mus spretus mice or cumulus cells of Mus musculus domesticus mice were transferred into M. m. domesticus (B6SJLF1 and B6D2F1) oocytes to observe parthenogenetic development through the morula stage. All B6D2F1 oocytes injected with somatic cytoplasm or mitochondria showed delayed development when compared to oocytes injected with buffer. The developmental rates were not different among injected cell sources, with the exception of testis-derived donor cells injected into B6SJLF1 oocytes (P < 0.01). The developmental rate of B6D2F1 oocytes injected with buffer alone (98.8% survival) was different from those injected with somatic cytoplasm (60.8% survival) or somatic mitochondria (56.5% survival) (P < 0.01). Conversely, injection of ooplasm into B6D2F1 oocytes did not affect parthenogenetic development (100% survival). Our results indicate that injection of somatic cytoplasm or mitochondria affected parthenogenetic development of murine oocytes. These results have further implications for in vitro fertilization protocols employing ooplasmic transfer where primary oocyte failure is not confirmed.

Animals↗

PubMed2004

Transgenic mouse with human mutant p53 expression in the prostate epithelium.

BACKGROUND: Apoptosis is disrupted in prostate tumor cells, conferring a survival advantage. p53 is a nuclear protein believed to regulate cancer progression, in part by inducing apoptosis. To test this possibility in future studies, the objective of the present study was to generate a transgenic mouse model expressing mutant p53 in the prostate (PR). METHODS: Transgene incorporation was tested using Southern analysis. Expression of mutant p53 protein was examined using immunofluorescence microscopy. Apoptosis in the PR was evaluated using the Tunnel method. RESULTS: A construct, consisting of the rat probasin promoter and a mutant human p53 fragment, was prepared and used to generate transgenic mice. rPB-mutant p53 transgene incorporation, as well as nuclear accumulation of mutant human p53 protein, was demonstrated. Prostatic intraepithelial neoplasia (PIN) III and IV were found in PR of 52-week old transgenic mice, whereas no pathological changes were found in the other organs examined. PR ability to undergo apoptosis following castration was reduced in rPB-mutant p53 mice as compared to non transgenic littermates. CONCLUSIONS: Transgenic rPB-mutant p53 mice accumulate mutant p53 protein in PR, resulting in neoplastic lesions and reduced apoptotic potential in the PR. Breeding rPB-mutant p53 mice with mice expressing an oncogene in their PR will be useful in examining interactions of multiple genes that result in progression of slow growing prostate tumors expressing oncogenes alone to metastatic cancer.

Animals↗

PubMed2004

Production of homoplasmic xenomitochondrial mice.

The unique features of mtDNA, together with the lack of a wide range of mouse cell mtDNA mutants, have hampered the creation of mtDNA mutant mice. To overcome these barriers mitochondrial defects were created by introducing mitochondria from different mouse species into Mus musculus domesticus (Mm) mtDNA-less (rho(0)) L cells. Introduction of the closely related Mus spretus (Ms) or the more divergent Mus dunni (Md) mitochondria resulted in xenocybrids exhibiting grossly normal respiratory function, but mild metabolic deficiencies, with 2- and 2.5-fold increases in lactate production compared with controls. The transfer of this model from in vitro to in vivo studies was achieved by introducing Ms and Md mitochondria into rhodamine-6G-treated Mm mouse embryonic stem (ES) cells. The resultant xenocybrid ES cells remained pluripotent, and live-born chimerae were produced from both Ms and Md xenocybrid ES cells. Founder chimeric females (G(0)) were mated with successful germ-line transmission of Ms or Md mtDNA to homoplasmic G(1) offspring. These xenocybrid models represent the first viable transmitochondrial mice with homoplasmic replacement of endogenous mtDNA and confirm the feasibility of producing mitochondrial defects in mice by using a xenomitochondrial approach.

Animals↗

PubMed2004

Analysis of lymphocyte development and function using the RAG-deficient blastocyst complementation system.

The RAG-deficient blastocyst complementation system (RBCS) represents a flexible and rapid method for the genetic analysis of lymphocyte function using a gene-targeting approach. In chimeras derived from manipulated embryonic stem cells injected into VDJ recombination-incapable, RAG-deficient blastocysts, any lymphoid cells past the prolymphocytic stage will be embryonic stem cell-derived. This approach can therefore bypass pitfalls such as pleiotropy and embryonic lethality to allow the analysis of targeted gene mutations with respect to lymphocyte development and function in a genetically uniform cell population. Thanks to recent advances in targeting techniques and in mouse embryo manipulation, this remarkably efficient technique has become a highly feasible and useful addition to any immunology research program. In this review, we discuss the technical aspects of the procedure, as well as its advantages and drawbacks compared to alternative approaches, and our practical experience in establishing the system at the University of Rochester.

Animals↗

PubMed2003

Functional respiratory chain analyses in murid xenomitochondrial cybrids expose coevolutionary constraints of cytochrome b and nuclear subunits of complex III.

The large number of extant Muridae species provides the opportunity of investigating functional limits of nuclear/mitochondrial respiratory chain (RC) subunit interactions by introducing mitochondrial genomes from progressively more divergent species into Mus musculus domesticus mtDNA-less (rho0) cells. We created a panel of such xenomitochondrial cybrids, using as mitochondrial donors cells from six murid species with divergence from M. m. domesticus estimated at 2 to 12 Myr before present. Species used were Mus spretus, Mus caroli, Mus dunni, Mus pahari, Otomys irroratus, and Rattus norvegicus. Parsimony analysis of partial mtDNA sequences showed agreement with previous molecular phylogenies, with the exception that Otomys did not nest within the murinae as suggested by some recent nuclear gene analyses. Cellular production of lactate, a sensitive indicator of decreased respiratory chain ATP production, correlated with divergence. Functional characterization of the chimeric RC complexes in isolated mitochondria using enzymological analyses demonstrated varying decreases in activities of complexes I, III, and IV, which have subunits encoded in both mitochondrial and nuclear genomes. Complex III showed a striking decline in electron transfer function in the most divergent xenocybrids, being greatly reduced in the Rattus xenocybrid and virtually absent in the Otomys xenocybrid. This suggests that nuclear subunits interacting with cytochrome b face the greatest constraints in the coevolution of murid RC subunits. We sequenced the cytochrome b gene from the species used to identify potential amino acid substitutions involved in such interactions. The greater sensitivity of complex III to xenocybrid dysfunction may result from the encoding of redox center apoproteins in both nuclear and mitochondrial genomes, a unique feature of this RC complex.

Adenosine Triphosphate↗

PubMed2003

Transgenic animal technology: alternatives in genotyping and phenotyping.

Over the past decade, breakthrough technologies in transgenic animal technology and functional genomics have played a central role in the explosive growth of rodent modeling and in scientific innovation. Various noninvasive alternatives to routine surgical biopsy have been described for genotypic and phenotypic analyses of laboratory animals. A number of options are available to refine or replace potentially painful and invasive procedures ranging from tissue biopsies (including tail biopsies and toe docking) to several blood sampling techniques. Unfortunately, adoption of many non- or minimally invasive alternatives has proven difficult on a number of fronts ranging from historical reservations to procedural expectations and actual experimental productivity. Similarly, a variety of phenotyping considerations have addressed throughput efficiencies and the health and well being of research animals. From an animal welfare perspective, marked increases in laboratory animal populations have accompanied rapid advancements spanning the life sciences. As described for rodent modeling, but with applications across many laboratory animal species, diverse procedural refinements are available that will readily aid in the analysis of whole animal models. Ultimately, non-invasive technologies and complementary refinements have bearing on the quality and reproducibility of data that are reported, as well as of critical importance to the well being and ethical management of animals at all developmental stages: from fetal existence, to the neonatal period, and on through adulthood.

Animal Use Alternatives↗

PubMed2003

Mouse embryo yield and viability after euthanasia by CO2 inhalation or cervical dislocation.

Efficient production of transgenic mice requires high yields of viable, healthy embryos. Cervical dislocation (without prior anesthesia) rather than CO2 inhalation as a means of euthanasia has been justified on the basis of the increased yield of viable ova, but controlled studies have not directly supported this contention. The American Veterinary Medical Association (AVMA) and Canadian Council on Animal Care (CCAC) Guides, and respective Institutional Animal Care and Use Committees (IACUC) have supported the use of CO2 as a preferred, humane method. The study reported here was undertaken to determine the relative yields of viable embryos from mice euthanized either by inhalation of 100% CO2 or by cervical dislocation. Inbred and hybrid mouse strains, representative of common strains used in genetic engineering experimentation included C57BL/6, FVB/N, and B6SJLF1. There was no difference in the embryo yields in comparisons using the two methods of euthanasia (P = 0.534). Decisions regarding the method of euthanasia can be made on the basis of criteria other than those associated with embryo yield and viability.

Administration, Inhalation↗

PubMed2002

Production of transmitochondrial mice.

With the advancement of various gene transfer technologies, the establishment of mitochondria transfer as a viable technique to genetically engineer mouse models paradoxically lagged behind other genetic technologies. The lack of demonstrable recombination in mtDNA necessitates different approaches to conventional transgenesis-based techniques. Initially, heteroplasmic mice were created to explore disease pathogenesis and mitochondrial dynamics in an in vivo system. Ultimately, transmitochondrial mouse models will be used to explore the role of the mitochondrial genome in human disease processes and in the development of novel human gene therapies. Here, we describe methodology to produce transmitochondrial mice (both homoplasmic and heteroplasmic models) harboring foreign mitochondrial genomes, using both embryo microinjection and embryonic stem (ES) cell-based approaches. Specific modeling and the procedures for mitochondrial transfer will be of considerable importance toward our understanding of discrete mitochondrial mutations, as well as lead to the development of novel strategies and therapies for human diseases influenced by mitochondrial DNA mutations.

Animals↗

PubMed2002

Mitochondrial activity in response to serum starvation in bovine (Bos taurus) cell culture.

In nuclear transfer procedures, in addition to nuclei, donor cell mitochondria are routinely transferred into recipient oocytes, and mitochondrial heteroplasmy has been reported. However, various protocols have resulted in either homoplasmy for recipient oocyte mitochondria or varying heteroplasmic levels in cloned animals. In nuclear transfer protocols, donor cells are subjected to serum-starvation prior to electroporation. Therefore, the relationship between culture conditions and mitochondrial activity was explored. Fibroblast cell lines were propagated from bovine ear epithelium, skin, skeletal muscle, or cumulus cells. In vitro mitochondrial viability was assessed in proliferative and confluent cells, cultured under serum-starvation or supplemented conditions. Cells were stained with MitoTracker Red CMXRos and comparative fluorescence intensities were assessed. The mitochondrial activity per cell was highest under proliferation, significantly lower at confluency (p < 0.001), and remained depressed after serum starvation for within a week (p < 0.001). Serum starvation induced an increase in mitochondrial viability in confluent cells. These results demonstrate that mitochondrial viability is dramatically affected by cell culture conditions. Consequently, specific cell culture parameters provide one explanation for the varying incidence of heteroplasmy identified in cloned animals. Future research should reveal whether specific cell culture parameters represent one of the factors for the varying incidence of heteroplasmy identified in cloned animals.

Animals↗