Mouse &lt;em&gt;in Utero&lt;/em&gt; Electroporation: Controlled Spatiotemporal Gene Transfection

Matsui, Asuka; Yoshida, Aya; Kubota, Mayumi; Ogawa, Michio; Shimogori, Tomomi

doi:10.3791/3024-v

Cited by 8 publications

(4 citation statements)

References 1 publication

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“…(i) Although the area plots can be obtained from any mouse line, a tomato allele needs to be bred with the mice under investigation in order to collect intensity data. To avoid breeding the tomato expressing transgene, transient embryo transfection could be performed within the uterus with an adenovirus carrying the reporter gene [ 48 ].…”

Section: Discussionmentioning

confidence: 99%

Imaging the dynamics of murine uterine contractions in early pregnancy

Dawson,

Flores,

Zou

et al. 2024

Biology of Reproduction

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Uterine muscle contractility is essential for reproductive processes including sperm and embryo transport, and during the uterine cycle to remove menstrual effluent. Even still, uterine contractions have primarily been studied in the context of preterm labor. This is partly due to a lack of methods for studying the uterine muscle contractility in the intact organ. Here, we describe an imaging-based method to evaluate mouse uterine contractility of both the longitudinal and circular muscles in the cycling stages and in early pregnancy. By transforming the image-based data into 3D spatiotemporal contractility maps, we calculate waveform characteristics of muscle contractions, including amplitude, frequency, wavelength, and velocity. We report that the native organ is highly contractile during the progesterone-dominant diestrus stage of the cycle when compared to the estrogen-dominant proestrus and estrus stages. We also observed that during the first phase of uterine embryo movement when clustered embryos move towards the middle of the uterine horn, contractions are dynamic and non-uniform between different segments of the uterine horn. In the second phase of embryo movement, contractions are more uniform and rhythmic throughout the uterine horn. Finally, in Lpar3-/- uteri, that display faster embryo movement, we observe global and regional increases in contractility. Our method provides a means to understand the wave characteristics of uterine smooth muscle in response to modulators and in genetic mutants. Better understanding uterine contractility in the early pregnancy stages is critical for the advancement of artificial reproductive technologies and a possibility of modulating embryo movement during clinical embryo transfers.

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Section: Discussionmentioning

confidence: 99%

Imaging the dynamics of murine uterine contractions in early pregnancy

Dawson,

Flores,

Zou

et al. 2024

Biology of Reproduction

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“…Mouse embryos were electroporated at E13.5 as previously described [24, 25]. The five mouse genes ORFs, were cloned into the polylinker (LinkerA) site of a pCAGGS-LinkerA- ires -nls-GFP backbone vector (pCAG).…”

Section: Materials Patients and Methodsmentioning

confidence: 99%

“…Embryo brains were recovered at E17.5 and processed as previously described [24, 25]. Immunohistochemistry and In situ hybridization experiments were performed on 16 µm or 20 µm thick Coronal sections, respectively [26].…”

Section: Materials Patients and Methodsmentioning

confidence: 99%

Slitrk/LAR-RPTP and disease-associated variants control neuronal migration in the developing mouse cortex independently of synaptic organizer activity

Medvedeva,

Billuart,

Jeanmart

et al. 2023

Preprint

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Slitrks and their ligands LAR-RPTPs are type I transmembrane proteins previously implicated in the etiology of various neuropsychiatric disorders including obsessive-compulsive disorders (OCDs) and schizophrenia. Over the last decade, their functions were extensively studied in hippocampal neurons in vitro and shown to shape synapse organization. Although both protein families are highly expressed prior to synapse formation, their function in earlier steps of cerebral cortex development remains unknown. Here we investigated the role of Slitrk1, Slitrk2, Slitrk3 and LAR-RPTPs (Ptprs and Ptprd) in the embryonic mouse cortex by acute genetic manipulation using in utero electroporation. All genes, except Slitrk3, promoted specific alterations in radial migration of glutamatergic neurons. Slitrk1 and Slitrk2 overexpression was associated with accumulation of neurons in distinct regions of the cortical plate. Using deletion mutants and a series of Slitrk variants associated with neurodevelopmental disorders (NDDs), we showed that distinct domains are crucial for intracellular Slitrk1 distribution and/or density and shape of VAMP2+ presynaptic boutons. Interestingly, bouton alterations did not correlate with the observed migration delays, suggesting that Slitrk1 influence cell migration independently on its synaptogenic function. Furthermore, co-electroporation experiments with LAR-RPTPs, mimicking their co-expression observed by scRNAseq, rescued the migration deficits, suggesting possible cis-interactions between Slitrks and LAR-RPTPs. Together, these data indicate that in the embryonic cerebral cortex Slitrks and LAR-RPTPs cooperate in consecutive steps of radial migration through distinct mechanisms than in synapse organization and support a relevant role of Slitrk/LAR-RPTP dysfunctions in NDDs at earlier stages of cortical development.

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“…Gene transfer by in utero electroporation into developing cortical cells in mice is efficient and transgene expression can persist for months in numerous cerebral targets, presenting several spatial and temporal advantages over other methods to study brain development in vivo . 7 , 70 , 71 , 72 , 73 , 74 , 75 However, efficient control of expression of transgenes after in vivo electroporation can be ameliorated using protocols based on a transposable-mediated gene expression switch, such as. 41 Note: Highly autofluorescent cell types in the samples may be detected as a false, very low positive signal (lower than the actual signal) even in the FMO/single- and non-transfected samples.…”

Section: Limitationsmentioning

confidence: 99%

Protocol for differential multi-omic analyses of distinct cell types in the mouse cerebral cortex

Meka,

Richter,

Rücker

et al. 2024

STAR Protocols

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Mouse <em>in Utero</em> Electroporation: Controlled Spatiotemporal Gene Transfection

Cited by 8 publications

References 1 publication

Imaging the dynamics of murine uterine contractions in early pregnancy

Imaging the dynamics of murine uterine contractions in early pregnancy

Slitrk/LAR-RPTP and disease-associated variants control neuronal migration in the developing mouse cortex independently of synaptic organizer activity

Protocol for differential multi-omic analyses of distinct cell types in the mouse cerebral cortex

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