A high-speed congenic strategy using first-wave male germ cells

Abstract

Background: 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). Even with marker-assisted selection, the so-called 'speed congenic strategy', it takes more than a year at N4 or N5.

Methodology/principal findings: Here we describe a new high-speed congenic system using round spermatids retrieved from immature males (22-25 days of age). We applied the technique to three genetically modified strains of mice: transgenic (TG), knockin (KI) and N-ethyl-N-nitrosourea (ENU)-induced mutants. The donor mice had mixed genetic backgrounds of C57BL/6 (B6):DBA/2 or B6:129 strains. At each generation, males used for backcrossing were selected based on polymorphic marker analysis and their round spermatids were injected into B6 strain oocytes. Backcrossing was repeated until N4 or N5. For the TG and ENU-mutant strains, the N5 generation was achieved on days 188 and 190 and the proportion of B6-homozygous loci was 100% (74 markers) and 97.7% (172/176 markers), respectively. For the KI strain, N4 was achieved on day 151, all the 86 markers being B6-homozygous as early as on day 106 at N3. The carrier males at the final generation were all fertile and propagated the modified genes. Thus, three congenic strains were established through rapid generation turnover between 41 and 44 days.

Conclusions/significance: This new high-speed breeding strategy enables us to produce congenic strains within about half a year. It should provide the fastest protocol for precise definition of the phenotypic effects of genes of interest on desired genetic backgrounds.

Figure 1. Definition of the optimal male age for spermatid collection.

a) Representative photomicrograph of a cell suspension prepared from the testis of a male mouse at 24 days of age. Arrows indicate round spermatids, which are easily identified by a round nucleus and a high cytoplasmic/nuclear ratio. b) The proportion of round spermatids among testicular cells from 18 days to 24 days after birth. The percentages of round spermatids in testicular suspension increased consistently from days 18 to 24 (P<0.05 between groups). The cells were counted in two different males by two different operators. The horizontal bars indicate the average.

Figure 2. Time course of generation turnover and the rate of markers homozygous for the C57BL/6 (B6) type.

a) Vasa–Venus transgenic strain. All markers (n = 74) were homozygous for B6 at N5 on day 188. b) Ednra^EGFP/+ knockin strain. All markers (n = 86) were homozygous for B6 at N3 on day 106 and N4 offspring were obtained on day 151. c) ENU-induced Gdf5 mutant strain. The N5 generation was obtained on day 190 and was 97.7% (172/176) homozygous for B6. There were 74, 86 and 176 polymorphic markers, which identified the alleles for the C57BL/6Cr:DBA/2Cr, C57BL/6Cr:129 and C57BL/6J:DBA/2J strains, respectively. Each generation turnover was between 42 and 45 days: the age of the donor male plus the gestation period (20 days) minus the one-day overlap between them.