Abstract
BackgroundIn 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 FindingsHere 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/SignificanceThis 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.
Highlights
For nearly 30 years, the genetic manipulation of laboratory mice has contributed substantially to the development of many fields in medical research and mammalian biology
This often raises serious concerns, because transgenic (TG) or knockout mice are generated in strains that have historically been selected for the ease and convenience of generating the TG or knockout strain, rather than phenotypic characterization of the mutation itself
Round spermatids can be collected from 17-day-old males at the earliest and their genomes can support full term embryonic development after injection into oocytes using round spermatid injection (ROSI) [9]
Summary
For nearly 30 years, the genetic manipulation of laboratory mice has contributed substantially to the development of many fields in medical research and mammalian biology. Most embryonic stem (ES) cell lines used for knockout experiments are derived from the 129 strain This strain has significant biological limitations that interfere with the phenotypic analysis of a target mutation. Mouse round spermatids—the youngest haploid male germ cells—appear first at 17 days after birth and can be used for the production of offspring by round spermatid injection (ROSI) into oocytes [9] We applied this technique to the generation of congenic strains from mice with mixed genotypes bearing a transgene, a targeted KI gene or chemically induced mutant genes. Our high-speed congenic system would be very useful for the accelerated analysis of genes of interest under a defined genetic background
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
