In situ transgenesis methods such as viruses and electroporation can rapidly create somatic transgenic mice but lack control over copy number, zygosity, and locus specificity. Here we establish mosaic analysis by dual recombinase-mediated cassette exchange (MADR), which permits stable labeling of mutant cells expressing transgenic elements from precisely defined chromosomal loci. We provide a toolkit of MADR elements for combination labeling, inducible and reversible transgene manipulation, VCre recombinase expression, and transgenesis of human cells. Further, we demonstrate the versatility of MADR by creating glioma models with mixed reporter-identified zygosity or with ''personalized'' driver mutations from pediatric glioma. MADR is extensible to thousands of existing mouse lines, providing a flexible platform to democratize the generation of somatic mosaic mice.

Summary This protocol focuses on the cloning and stable integration of sequences of interest by the use of a mosaic analysis with dual recombinases (MADR) plasmid that includes fusion proteins or independent proteins under the control of 2A peptide or IRES elements. Additionally, we describe how to generate a neural stem cell culture from Gt(ROSA)26Sort m4(ACTB-tdTomato, EGFP)Luo/ J mice, and validate the MADR plasmids in vitro and in vivo by neonatal mouse brain electroporation. This protocol can be generalized to analyze any transgenic element using MADR technology. For complete details on the use and execution of this protocol, please refer to Kim et al. (2019) .

Background: Sperm selection procedures for future strategies that aim to select normal spermatozoa with intact DNA to improve intracytoplasmic sperm injection (ICSI) outcomes are in early developing stage. Objectives:The objective is to find out whether the sperm selection procedure based on the ability of spermatozoa to traverse the cumulus cells could improve clinical outcomes of ICSI technique in infertile couples with male factor etiology. Materials and methods:For this single-blind clinical trial, mature metaphase II oocytes were retrieved from 150 couples with male factor infertility, male age lower than 45 years and female age under 38 years. These couples were divided into two groups.In control group (n = 75), spermatozoa processed by density gradient centrifugation (DGC) were used to inject the oocytes. In the study group (n = 75), the oocytes were divided into sibling groups. In one sibling group (DGC), the oocytes were inseminated with DGC-processed spermatozoa while in the other group (DGC-CC), they were inseminated with DGC-processed spermatozoa that passed cumulus oophorous column.Results: Mean fertilization and embryo quality were significantly higher in DGC-CC group compared to DGC and control group. In addition, mean of chemical pregnancy (52.27% vs. 34.14%; p = 0.05), clinical pregnancy based on sac (52.27% vs. 32.92%; p = 0.03), clinical pregnancy with heart beat (52.27% vs. 25.60%; p = 0.003) and ongoing pregnancy (43.18% vs. 21.95%; p = 0.02) rates were significantly higher in DGC-CC group compared to control group. Conclusion:Sperm selection based on integrated systems such as DGC and ability to pass through cumulus oophorous column could improve clinical outcomes of ICSI in couples with male factor infertility.

Viral vectors and electroporation (EP)-mediated gene transfers are efficient means of inducing somatic mosaicismin mice, but they lack the exquisite control over transgene copy number, gene zygosity, and genomic-locus specificity that genetically engineered mouse models (GEMMs) provide. Here, we develop and demonstrate a simple and generalizable in vivo method, mosaic analysis by dual recombinase-mediated cassette exchange (MADR). MADR allows for stable labeling of mutant cells express transgenic elements from a precisely-defined chromosomal locus. To test our method, we generated reporter-labeled lineages from stem and progenitor cells in a well-defined Rosa26 mTmG mouse. We demonstrate the power and versatility of MADR by creating novel glioma models with mixed, reporter-defined zygosity or with "personalized" driver mutations from pediatric glioma-each manipulation altering the profile of resulting tumors. Thus, MADR provides a high-throughput genetic platform for the dissection of development and disease, and this rapid method can be applied to the thousands of existing gene-trap mice.

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