Northern blot | EPFL Graph Search

The northern blot, or RNA blot, is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression rates during differentiation and morphogenesis, as well as in abnormal or diseased conditions. Northern blotting involves the use of electrophoresis to separate RNA samples by size, and detection with a hybridization probe complementary to part of or the entire target sequence. Strictly speaking, the term 'northern blot' refers specifically to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane. However, the entire process is commonly referred to as northern blotting. The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University. Northern blotting takes its name from its simi

Studies on lentiviral-mediated transgenesis

Marc-Olivier Sauvain

Transgenic animals are essential research tools, whether to address basic biological questions or to develop preclinical models of human diseases. Their generation through the injection of naked plasmid DNA into the male pronucleus of a fertilized oocyte has been the standard practice for almost 3 decades. However, this approach is invasive, largely limited to the mouse and results in low rates of transgene integration. Although the inefficiency of pronuclear injection can be overcome in small animals by high-throughput screening for transgene integration, this becomes economically more challenging in larger animals, such as sheep, pigs or cows, because of the extreme costs associated with this procedure (

60'000-

300'000). Thus, new strategies to enhance the production and the variety of transgenic animals would be an important asset. One such approach has emerged through the use of lentiviral vectors, a gene delivery system that can efficiently transfer and stably integrate its cargo into a wide variety of cell types from numerous species, and has been successfully applied to generate transgenic mice, rats, pigs and cows. The increasing use of lentivector-mediated transgenesis warrants a full analysis of its modalities. As a step towards this goal, the present study aimed at characterizing the genotypic features of transgenic mice generated by lentiviral infection of zygotes. First, as an indirect method to measure the kinetics of lentiviral integration in early embryos, we examined the rates of transmission of individual proviruses from G0 transgenic mice to their G1 progeny using a PCR-based technique specific for each integrant. The mean rate of transmission of 44% for individual proviruses suggests that vector integration was most often established between the post-S phase of the one-cell stage and pre-S phase of the two-cell stage. We then moved on to define proviral integration site selection in lentivector-generated transgenic mice. Using LAM-PCR-mediated amplification and sequencing of the host genome-provirus junctions, we found that the frequency of proviral integration inside a gene was 1.75- and 1.88-fold higher in transgenic mice and 3T3 fibroblasts, respectively, than expected from the distribution of annotated genes in the mouse genome (Pmice .03e 09; P3T3 5.55e 16). Moreover, integration was not influenced by the transcriptional orientation of the target gene and tended to favour the middle of transcribed regions. We also observed a subtle difference in the integration patterns of lentiviral vectors in 3T3 fibroblasts and transgenic mice, with the latter exhibiting a slightly higher frequency of integration into repeats. Although this difference was statistically not significant, it might reflect the elevated transcriptional activity of repetitive elements in preimplantation embryos. Since proviruses favour intragenic localization, we analyzed G2 mice homozygous for specific lentiviral integrants using PCR-based techniques and observed that the frequency of homozygosity was below the expected mendelian transmission rate of 25%. This suggested that some of the homozygous G2 mice must be nonviable due to the potential of retrovirus integrants to inactivate target genes. However, the normal rates of G1 representation of proviruses that did not achieve homozygosity in G2 suggested an absence of toxicity in the heterozygous state. Processes governing the earliest steps of mammalian development are still incompletely understood. In particular, the stage at and modalities by which early blastomeres become committed to either the inner cell mass (ICM), which subsequently yields the embryo proper, or the trophectoderm, which serves as precursor of extra-embryonic tissues, is unclear. The long held "random" hypothesis for development of the embryonic-abembryonic axis states that, within these two structures, early blastomeres are equally represented. However, recent lineage-tracing experiments support a model of "developmental bias", in which the progeny of the early blastomeres is allocated in a biased fashion. In our study, we took advantage of the specific integration pattern of lentiviral vector – i.e. each lentiviral vector integrates the host genome at specific sites – to "fingerprint" individual blastomeres and to follow them during embryogenesis. For this, we injected lentiviral vectors into embryos at either the one-cell or the two-cell stage. The Southern Blot (SB) analysis performed at E12.5 revealed that the infection at the one-cell stage yielded integrants shared for their majority by both embryonic and extra-embryonic tissues (74% of similar bands), while the infection at the two-cell stage induced a drastically different picture, with only a minority of common integrants between both types of tissues (22% of similar bands). This suggests that early blastomeres – from the two-cell stage on – contribute differentially to these lineages, which argues against a purely stochastic model. However, we still obtained 22% of shared integrations after infection at the two-cell stage, with only 8 out of 27 embryos showing a complete segregation of the proviral integration patterns. This rules out a "fully determined" model. Thus, our observations rather support the "developmental bias" hypothesis.

EPFL2009

MicroRNA profiling by simultaneous selective isotachophoresis and hybridization with molecular beacons

Alexandre Louis André Persat

We present and demonstrate a novel assay for the detection and quantification of microRNA (miRNA) that leverages isotachophoresis (ITP) and molecular beacon (MB) hybridization. We use ITP to selectively preconcentrate miRNA from total RNA. We simultaneously focus MBs and use the ITP zone as a 10 pL reactor with active mixing where MBs fluoresce upon hybridization to target miRNA. To increase both sensitivity and selectivity, we leverage a multistage ITP strategy composed of three discrete regions of different concentrations of denaturant, sieving matrix, and magnesium chloride. We show that ITP hybridization is specific and selective to the miRNA target. We demonstrate ITP hybridization of miRNA in a biologically relevant case by detecting and quantifying miR-122 in human kidney and liver. ITP hybridization is a cheap, simple, high-speed, and amplification-free miRNA profiling method which requires small amounts (order 100 ng) of sample. The technique therefore represents an attractive alternative to PCR or Northern blot for miRNAs.

American Chemical Society2011

Opposite regulation of calbindin and calretinin expression by brain-derived neurotrophic factor in cortical neurons

Pierre Magistretti, Jean-Luc Martin

Regulation of calbindin and calretinin expression by brain-derived neurotrophic factor (BDNF) was examined in primary cultures of cortical neurons using immunocytochemistry and northern blot analysis. Here we report that regulation of calretinin expression by BDNF is in marked contrast to that of calbindin. Indeed, chronic exposure of cultured cortical neurons for 5 days to increasing concentrations of BDNF (0.1-10 ng/ml) resulted in a concentration-dependent decrease in the number of calretinin-positive neurons and a concentration-dependent increase in the number of calbindin-immunoreactive neurons. Consistent with the immunocytochemical analysis, BDNF reduced calretinin mRNA levels and up-regulated calbindin mRNA expression, providing evidence that modifications in gene expression accounted for the changes in the number of calretinin- and calbindin-containing neurons. Among other members of the neurotrophin family, neurotrophin-4 (NT-4), which also acts by activating tyrosine kinase TrkB receptors, exerted effects comparable to those of BDNF, whereas nerve growth factor (NGF) was ineffective. As for BDNF and NT-4, incubation of cortical neurons with neurotrophin-3 (NT-3) also led to a decrease in calretinin expression. However, in contrast to BDNF and NT-4, NT-3 did not affect calbindin expression. Double-labeling experiments evidenced that calretinin- and calbindin-containing neurons belong to distinct neuronal subpopulations, suggesting that BDNF and NT-4 exert opposite effects according to the neurochemical phenotype of the target cell.

2000

Studies on lentiviral-mediated transgenesis

Marc-Olivier Sauvain

60'000-

EPFL2009