Microinjection | EPFL Graph Search

Microinjection is the use of a glass micropipette to inject a liquid substance at a microscopic or borderline macroscopic level. The target is often a living cell but may also include intercellular space. Microinjection is a simple mechanical process usually involving an inverted microscope with a magnification power of around 200x (though sometimes it is performed using a dissecting stereo microscope at 40–50x or a traditional compound upright microscope at similar power to an inverted model). For processes such as cellular or pronuclear injection the target cell is positioned under the microscope and two micromanipulators—one holding the pipette and one holding a microcapillary needle usually between 0.5 and 5 μm in diameter (larger if injecting stem cells into an embryo)—are used to penetrate the cell membrane and/or the nuclear envelope. In this way the process can be used to introduce a vector into a single cell. Microinjection can also be used in the cloning of organisms, in the study of cell biology and viruses, and for treating male subfertility through intracytoplasmic sperm injection (ICSI, ˈɪksi ). The use of microinjection as a biological procedure began in the early twentieth century, although even through the 1970s it was not commonly used. By the 1990s, its use had escalated significantly and it is now considered a common laboratory technique, along with vesicle fusion, electroporation, chemical transfection, and viral transduction, for introducing a small amount of a substance into a small target. There are two basic types of microinjection systems. The first is called a constant flow system and the second is called a pulsed flow system. In a constant flow system, which is relatively simple and inexpensive though clumsy and outdated, a constant flow of a sample is delivered from a micropipette and the amount of the sample which is injected is determined by how long the needle remains in the cell. This system typically requires a regulated pressure source, a capillary holder, and either a coarse or a fine micromanipulator.

Intestinal organoid cocultures with microbes

Devanjali Dutta

This protocol comprises various methods to coculture organoids (particularly human small intestinal and colon organoids) with microbes, including microinjection into the lumen and periphery of 3D organoids and exposure of organoids to microbes in a 2D layer. Adult-stem-cell-derived organoids model human epithelial tissues ex vivo, which enables the study of host-microbe interactions with great experimental control. This protocol comprises methods to coculture organoids with microbes, particularly focusing on human small intestinal and colon organoids exposed to individual bacterial species. Microinjection into the lumen and periphery of 3D organoids is discussed, as well as exposure of organoids to microbes in a 2D layer. We provide detailed protocols for characterizing the coculture with regard to bacterial and organoid cell viability and growth kinetics. Spatial relationships can be studied by fluorescence live microscopy, as well as scanning electron microscopy. Finally, we discuss considerations for assessing the impact of bacteria on gene expression and mutations through RNA and DNA sequencing. This protocol requires equipment for standard mammalian tissue culture, or bacterial or viral culture, as well as a microinjection device.

NATURE PORTFOLIO2021

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

Studies on lentiviral-mediated transgenesis

Marc-Olivier Sauvain

60'000-

EPFL2009