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Video protocol

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification
JoVE 3155 11/18/2011

BRC Current Technology Oct 2011

2. High-Speed Congenics Using First-Wave
Male Germ Cells

The fastest generation turnover in mammals
(40-45 days)

BRC Current Technology(pdf)PDF: 115KB



Immature sperm (round spermatids) in the dotted circle (right) from a 22-day old male (black mouse) can be used in high-speed breeding of genetically mouse models


Time course of generation turnover and the rate of markers for the expected genetic background. With immature sperm, all markers became homozygous at 180 days in this case.

In laboratory mice and rats, congenic breeding is essential for analyzing the genes of interest on specific genetic
backgrounds and for analyzing quantitative trait loci. However, in theory it takes about 3-4 years to achieve a
strain carrying about 99% of the recipient genome at the tenth backcrossing (N10). The recently developed‘speed
congenic strategy’ using marker-assisted selection has shortened the required generations to N4 or N5 but it
still takes more than a year. To accelerate the congenic breeding, we examined the feasibility of the
microinsemination (sperm injection) technique using the “first wave” sperm cells from immature males. Haploid
germ cells (round spermatids) first appear in immature male mice at 18 days after birth and their number
increases to the practical level for microinsemination at 22-25 days [1]. The technique was then applied to the
congenic strategy in combination with marker-assisted selection. Transgenic, knock-in, and ENU-mutant strains
with mixed genetic backgrounds eventually became C57BL/6 congenic strains according to the polymorphic
markers used (n= 74 to 176) [2]. The strategy was proven to be effective for other genetic backgrounds (e.g.,
NOD-scid). It should provide the fastest protocol for precise definition of the phenotypic effects of genes of
interest on desired genetic backgrounds.

Collaborated with Dr. H. Kurihara (Graduate School of Medicine, University of Tokyo, Japan)

RIKEN Research :


1. Miki et al., J Reprod Dev 50: 131-137 (2004)
2. Ogonuki et al., PLoS One 4: e4943 doi:10.1371/journal.pone.0004943 (2009)