Analysis of mouse EphA knockins and knockouts suggests that retinal axons programme target cells to form ordered retinotopic maps

I present a novel analysis of abnormal retinocollicular maps in mice in which the distribution of EphA receptors over the retina has been modified by knockin and/or knockout of these receptor types. My analysis shows that in all these cases, whereas the maps themselves are discontinuous, the graded distribution of EphA over the nasotemporal axis of the retina is recreated within the pattern of axonal terminations across rostrocaudal colliculus. This suggests that the guiding principle behind the formation of ordered maps of nerve connections between vertebrate retina and superior colliculus, or optic tectum, is that axons carrying similar amounts of Eph receptor terminate near to one another on the target structure. I show how the previously proposed marker induction model embodies this principle and predicts these results. I then describe a new version of the model in which the properties of the markers, or labels, are based on those of the Eph receptors and their associated ligands, the ephrins. I present new simulation results, showing the development of maps between two-dimensional structures, exploring the role of counter-gradients of labels across the target and confirming that the model reproduces the retinocollicular maps found in EphA knockin/knockout mice. I predict that abnormal distributions of label within the retina lead to abnormal distributions of label over the target, so that in each of the types of knockin/knockout mice analysed, there will be a different distribution of labels over the target structure. This mechanism could be responsible for the flexibility with which neurons reorganise their connections during development and the degree of precision in the final map. Activity-based mechanisms would play a role only at a later stage of development to remove the overlap between individual retinal projection fields, such as in the development of patterns of ocular dominance stripes.     

Facile methods for generating human somatic cell gene knockouts using recombinant adeno-associated viruses

Emerging evidence suggests that recombinant adeno-associated viral (rAAV) vectors can be used for specific gene targeting in human somatic cells. We have developed an rAAV vector construction procedure employing fusion PCR and a single cloning step that considerably simplifies the knockout process. We demonstrate its utility by disrupting genes at specific positions within human colon cancer cells as well as within immortalized normal epithelial cells. This technology should be broadly applicable to in vitro studies that require the manipulation of the human genome.     

Spontaneous chromosome aberrations in human somatic cells

Conclusion In conclusion it is necessary to say that at present, we cannot consider whether there may be a geographical difference in frequency of spontaneous chromosome aberrations in somatic cells. For that purpose, a very abundant experimental material is required, as well as an improvement in methodology: what causes the difference in the results of various investigators; what methodical principles should be used for collection of data.Important factors in the differences of frequencies of spontaneous chromosome aberrations are likely to be the conditions of cultivation and making the preparations, as well as the methods of scoring the chromosome aberrations. The international standardisation of the cultivation conditions and of the estimates of chromosome aberrations is needed for the further study of the rate and reasons of the spontaneous mutation process in somatic cells.     

Radiation induced inversions in human somatic cells

Peri- and paracentric inversions induced by various types of ionizing radiation (gamma and alpha-rays, neutron and neon beams) are analysed. Their frequencies significantly increase for radiation doses greater than or equal to .5 Gy. Their distribution does not seem to be at random. Pericentric are detected 3 to 4 times more frequently than paracentric inversions. Some identical inversions are recurrently induced. A proportion reproduces inversions detected in human cytogenetics laboratories and a larger proportion, chromosomes of other primate species. It seems that breakages, which numbers are roughly proportional to chromosome lengths, lead to reassociations with a limited number of combinations.     

Genetic analysis of homologous DNA recombination in vertebrate somatic cells

The maintenance of genomic stability and the ability to repair induced DNA damage in vertebrate cells require homologues of the yeast RAD52 epistasis group genes. The homologous recombination carried out by the products of these genes is essential and appears to be closely linked to DNA replication. Defects in recombination and associated activities are implicated in human cancer. This review summarises recent biochemical and genetic findings on the roles played by the vertebrate RAD52 group gene products in recombination. We describe the phenotypic analysis of genetically engineered mammalian and chicken mutants of homologous recombination genes.     

Genetic correlations of fighting ability with somatic cells and longevity in cattle

The success in competitions may be stressful for animals and costly in terms of immune functions and longevity. Focusing on Aosta Chestnut and Aosta Black Pied cattle, selected for their fighting ability in traditional competitions, this study investigated the genetic relationships of fighting ability with udder health traits (somatic cell score and two threshold traits for somatic cells), longevity (length of productive life and number of calvings) and test-day milk, fat and protein yield. Herdbook information and phenotypic records that have been routinely collected for breeding programs in 16 years were used for the abovementioned traits. Data belonged to 9328 cows and 19 283 animals in pedigree. Single-trait animal model analyses were run using a Gibbs sampling algorithm to estimate the variance components of traits, and bivariate analyses were then performed to estimate the genetic correlations. Moderate positive genetic correlations (r_a) were found for fighting ability with somatic cell score (r_a=0.255), suggesting that greater fighting ability is genetically related to a detriment in udder health, in agreement with the theory. The high positive genetic correlation between fighting ability and longevity (average r_a=0.669) suggests that the economic importance of fighting ability (the winning cows get an higher price at selling) had probably masked the true genetic covariances. The genetic correlation between milk yield traits and fighting ability showed large intervals, but the negative values (average r_a=−0.121) agreed with previous research. This study is one of the few empirical studies on genetic correlations for the competitive success v. immune functions and longevity traits. The knowledge of the genetic correlations among productive and functional traits of interest, including fighting ability, is important in animal breeding for a sustainable genetic improvement.     

植物体细胞原生质体遗传转化研究

重点介绍了植物体细胞原生质体遗传转化的方法和当前已经取得的成果,同时提出了目前原生质体遗传转化中存在的问题,展望了今后的工作重点。植物原生质体遗传转化的方法主要有：PEG介导转化法、电击穿孔转化法、脂质体介导转化法、农杆菌共培养转化法等。     

Transdifferentiation of human somatic cells by ribosome

Ribosomes are intracellular organelles ubiquitous in all organisms, which translate information from mRNAs to synthesize proteins. They are complex macromolecules composed of dozens of proteins and ribosomal RNAs. Other than translation, some ribosomal proteins also have side‐jobs called “Moonlighting” function. The majority of these moonlighting functions influence cancer progression, early development and differentiation. Recently, we discovered that ribosome is involved in the regulation of cellular transdifferentiation of human dermal fibroblasts (HDFs). In vitro incorporation of ribosomes into HDFs arrests cell proliferation and induces the formation of cell clusters, that differentiate into three germ layer derived cells upon induction by differentiation mediums. The discovery of ribosome induced transdifferentiation, that is not based on genetic modification, find new possibilities for the treatment of cancer and congenital diseases, as well as to understand early development and cellular lineage differentiation.     

Aneugenic effect of ionizing radiation in mammalian and human somatic cells

Aneuploidy is among the most serious impairments of hereditary material in somatic and germline cells of living organisms. Chromosome loss or the appearance of an extra homolog in the chromosome set can result in either cell death or the development of various neoplasms with high probability of malignancy. It was traditionally believed that ionizing radiation produces primarily a clastogenic effect. However, there is apparently an aneugenic component of radiation, with mechanism different from that of structural chromosome damage. The present review focuses on the evidence for the existence of the aneugenic effect of ionizing radiation in mammalian and human somatic cells.     

Genetic analysis of dexamethasone resistance in L cells by somatic cell hybridization

Stable dexamethasone resistant and receptor-containing (R+) variants of L cells have been characterized by somatic cell hybridization. Neither of the variants had a clearly dominant phenotype in hybrids with dexamethasone-sensitive fibroblast lines, i.e. the resistance of the variants was not due to transdominant factors. Somatic cell hybrids formed between one of the R+-resistant clones and an independent resistant fibroblast cell line showed complementation--the hybrid clones were as sensitive to the steroid as the sensitive parental lines. Complementation, however, disappeared after continued culture of the clones. The return of the dexamethasone-sensitive phenotype was not always linked with similar changes in the responsiveness to another steroid, e.g. progesterone. Our clones can be considered to be resistant variants, designated death-less (d-), where the cells are defective in a non-receptor component involved in the hormone response. The fact that complementation can occur indicates the existence of at least two such steps in the pathway.     

Genetic effects induced by nickel sulfate in germline and somatic cells of WR mice

We examined the effects of nickel sulfate at doses 0.5 to 5.0 mg/kg (LD50) on the frequency of dominant lethal mutations and two-strand DNA breaks (TSBs) in germline cells and on an increase in frequency in gene mutations W(y) in pigment cells of first-generation mice. The results indicated that spermatogenesis stages most sensitive to nickel sulfate (at a dose of 1.0 mg/kg) are spermatozoids, early spermatids, late spermatocytes, and stem spermatogonia. No statistically significant increase in the total TSB level was detected in spermatozoids 4 weeks after exposure. At the same time, a significant (P < 0.05) increase in percentage of cells with an extremely high level of DNA fragmentation (supposedly apoptotic cells) was observed upon exposure at a dose of 0.5 mg/kg. Nickel sulfate at doses of 5.0 and 1.0 mg/kg induced a marked increase in the c-kit gene expression in pigment cells of heterozygous first-generation WR mice as compared to control (P < 0.001). It was shown that the nonobservable adverse effect level (NOAEL) of nickel sulfate on the dominant lethal mutation frequency and gene mutations was 1/200 LD50, while the lowest observable adverse effect level (LOAEL) was 1/100 LD50.     

Use of gene knockouts in cultured cells to study apoptosis

The avian DT40 cell system represents a novel method to generate loss of function mutations in vertebrate cells. These chicken B lymphoma cells undergo homologous recombination at very high frequencies and can thus be used to "knock out" genes believed to function in apoptotic processes. The knockout cells can then be used to determine how the cell death process is modulated after induction of apoptosis and to order components in cell death pathways. The system can be further modified, using tetracycline-responsive promoters, to allow expression of wild-type cDNAs to rescue "knockout cells" if the gene of interest is essential. Alternatively, cDNA expression constructs containing mutations or deletions in the cDNA encoding the absent protein can be used to delineate functional domains. cDNA expression libraries or known proteins believed to function downstream of the target in a signal transduction pathway could also be transfected into the knockout cell line, and the resultant cells could be assayed for complementation and/or rescue of the apoptotic alteration/defect. Finally, the system has recently been adapted to allow disruption of human genes in DT40/human hybrid cell lines thereby potentially extending this system for use in studying human genes.     

The fate of human sperm-derived mtDNA in somatic cells.

Inheritance of animal mtDNA is almost exclusively maternal, most likely because sperm-derived mitochondria are actively eliminated from the ovum, either at or soon after fertilization. How such elimination occurs is currently unknown. We asked whether similar behavior could be detected in somatic cells, by following the fate of mitochondria and mtDNAs after entry of human sperm into transformed cells containing mitochondria but lacking endogenous mtDNAs (rho0 cells). We found that a high proportion (10%-20%) of cells contained functioning sperm mitochondria soon after sperm entry. However, under selective conditions permitting only the survival of cells harboring functional mtDNAs, only approximately 1/10(5) cells containing sperm mitochondria survived and proliferated. These data imply that mitochondria in sperm can enter somatic cells relatively easily, but they also suggest that mechanisms exist to eliminate sperm-derived mtDNA from somatic cells, mechanisms perhaps similar to those presumed to operate in the fertilized oocyte.