Double UP: A Dual Color, Internally Controlled Platform for in utero Knockdown or Overexpression

Abstract

In utero electroporation (IUE) is a powerful tool for testing the role of genes in neuronal migration and function, but this technique suffers from high degrees of variability. Such variability can result from inconsistent surgery, developmental gradients along both rostral-caudal and medial-lateral axes, differences within littermates and from one litter to another. Comparisons between control and experimental electroporations rely on section matching, which is inherently subjective. These sources of variability are cumulative, leading to difficult to interpret data and an increased risk of both false positives and false negatives. To address these limitations, we developed two tools: (1) a new plasmid, termed Double UP, which combines LoxP-flanked reporters and limiting Cre dosages to generate internal controls, and (2) an automated program for unbiased and precise quantification of migration. In concert, these tools allow for more rigorous and objective experiments, while decreasing the mice, time, and reagents required to complete studies.

RapID Cell Counter: Semi-Automated and Mid-Throughput Estimation of Cell Density within Diverse Cortical Layers

“…Further, we have demonstrated the flexibility of RapID to detect co-labeled cells with diverse morphologies. Although other automated cell counter methods exist (Smith et al, 2018), to our knowledge, only the recently published TRON program simultaneously quantifies cell abundance at spatial resolution and is freely available (Taylor et al, 2020). We attempted to compare RapID cell counts with TRON, which is optimized for use with an internally-controlled co-labeling IUE method, but were limited in our ability to apply the approach to our images due to a variety of factors, including stringent requirements for specific z-stacking parameters and a file format produced from proprietary software.…”

miR-137 and miR-122, two outer subventricular zone non-coding RNAs, regulate basal progenitor expansion and neuronal differentiation

“…Injected plasmids were: pUB6-GFP and pUB6-TOM (0,5 mg/mL); pSilencer-U6-scram, pSilencer-U6-miR-137 and pSilencer-U6-miR-122 (1 mg/mL); dUP-Cd63 and dUP-Myt1l (2 mg/mL, subcloned from double UP mClover to Scarlet, Addgene #125134, ( Taylor et al, 2020 )); DFRS, DFRS-137 and DFRS-122 (1 mg/mL, subcloned from DFRS, De Pietri Tonelli et al., 2006 ), pmiRGLO and pmirGLO + Cadm2 3′UTR and pmirGLO + Osbpl6 3′UTR and pmirGLO + Zfp68 3′UTR (40 ng/well).…”

“…As this gene is flanked by loxP sites, subsequent delivery of Cre protein results in the excision of mClover3 sequence, allowing for expression of mScarlet and the gene of interest (cloned at the MCS). A vector backbone (Double UP, Addgene #125134, Taylor et al., 2020 ) was used to allow for temporal control of gene expression ( CAG-loxP-mClover3-polyA-loxP-mScarlet-MCS-polyA ). Genes of interest were PCR amplified from a cDNA library from mouse embryonic brain RNA extracted at E14.5.…”

In Utero Electroporation for Manipulation of Specific Neuronal Populations

“…To further dissect the establishment and functions of neuronal networks, it is essential to understand the connectivity and interactions between multiple subsets of neuronal populations. A recent method called dual in utero electroporation serves as an effective tool to target multiple populations of neurons within the same circuit in a spatially and temporally specific manner [ 104 , 105 , 106 ]. This method has been developed to label or genetically manipulate different subtypes of neurons in different locations.…”