Generation and isolation of single cells from mouse brain with mosaic analysis with double markers-induced uniparental chromosome disomy

Abstract

Summary Mosaic analysis with double markers (MADM) technology enables concomitant fluorescent cell labeling and induction of uniparental chromosome disomy (UPD) with single-cell resolution. In UPD, imprinted genes are either overexpressed 2-fold or are not expressed. Here, the MADM platform is utilized to probe imprinting phenotypes at the transcriptional level. This protocol highlights major steps for the generation and isolation of projection neurons and astrocytes with MADM-induced UPD from mouse cerebral cortex for downstream single-cell and low-input sample RNA-sequencing experiments. For complete details on the use and execution of this protocol, please refer to Laukoter et al. (2020b) .

Genetic mosaic dissection of candidate genes in mice using mosaic analysis with double markers

“…MADM can also be applied for experiments involving flow cytometry. For details and a step-by-step protocol on the experimental workflow specific for flow cytometry experiments, we refer to ( Laukoter et al., 2020a ). Specific considerations when breeding experimental MADM animals are provided in the next two sections of the protocol:…”

“…MADM can also be applied for experiments involving flow cytometry. For details and a step-by-step protocol on the experimental workflow specific for flow cytometry experiments, we refer to ( Laukoter et al., 2020a ).…”

“…For more details on the MADM technology, please refer to Zong et al. (2005) , Beattie et al (2020) , Laukoter et al. (2020a) , and Contreras et al.…”

A genome-wide library of MADM mice for single-cell genetic mosaic analysis

“…MADM technology represents one approach to probe genomic imprinting and the function of imprinted genes (Hippenmeyer et al, 2013;Laukoter et al, 2020b). MADM can be applied to create uniparental chromosome disomy (UPD; somatic cells with two copies of either the maternal or paternal chromosome) and visualize imprinting effects at morphological and transcriptional levels with single-cell resolution (Hippenmeyer et al, 2013;Laukoter et al, 2020aLaukoter et al, , 2020c.…”

“…Single-cell analysis of imprinting phenotypes in UPD One MADM application includes the property to generate cells with UPD and thus enable the study of imprinting phenotypes at a single-cell level (Hippenmeyer et al, 2013;Laukoter et al, 2020aLaukoter et al, , 2020c. In fact, technical limitations so far only allowed the investigation of UPD at the whole-animal level but lacked the resolution to obtain insights at the cellular level (Pauler et al, 2021).…”