Experiment
The current study presents a thorough genetic dissection of the previously mapped QTL Led2 identified using the BcG66H-MMU1 strain (B6.SEG-(D1Mit81-rs6259837)) which harbored a high rate of embryonic lethality on chromosome 1, corresponding to a spretus fragment (SEG/Pas) carried by the BcG66H-MMU1 substrain [J:150137].
Fifteen substrains were created from BcG66H-MMU1 animals that encompassed distinct overlapping spretus (SEG/Pas) fragments. In vivo high frequency ultrasonography was used to follow embryonic development . Within the Led2 QTL a region of approximately 6 Mb, Led2a, was identified which had a main effect on the rate of embryonic death. A second region, Led2b, was also identified which could have a small effect on the phenotype, although it was not statistically demonstrated in this study.
The 15 new substrains were generated from backcrosses of BcG66H-MMU1 with C57BL/6J mice, each of them presenting a unique sub-fragment of the Led2 QTL. Eight new microsatellite markers located on MMU1 were genotyped to more precisely map the boundaries of the spretus fragment in the BcG66H-MMU1 genome. To follow the gestation in vivo, three ultrasonographic examinations were performed at three time points. At each examination the number of implanted embryos in each uterine horn as well as their status (alive or dead) were assessed. For each gestation, the embryonic lethality rate was calculated as the number of reabsorbed embryos in both horns reported to the total number of implanted embryos.
Among the 15 starting substrains, seven survived and were used in the study - recombinants R3, R4, R5, R6, R10, R13 and R14. QTL analysis was performed by genotype/phenotype segregation. Both R3 and R5 strains exhibited the phenotype and shared a large spretus region (>84.5 Mb to 90.0 Mb) with the R6 strain which did not display the phenotype.
The first spretus sub fragment was called Led2a and encompassed D1Mit50 to D1Mit305 (>84.5 Mb to 90.5 Mb) and a second region labeled Led2b was located at rs3692309 (>92.5 Mb to <100.3 Mb).
To statistically prove the presence of the two QTLs, Led2a and Led2b, each one responsible for a part of the effect on the phenotype, and an eventual epistatic interaction between the two able to increase embryonic death, several recombinants were compared among themselves, R6 bearing Led2a, R4 bearing Led2b and R3 (or R5) bearing both spretus regions.
Statistical results (significant difference at p</=0.01) proved the presence of the Led2a QTL when the mean rate of embryonic death was compared between R4 and R3 or R5. However, the comparison between R6 and R3 (or R5) did not statistically prove the presence of the Led2b QTL. The results did not support the presence of an epistatic interaction between the Led2a and Led2b regions, but a Led2b additive effect may be revealed by increasing the sample size of the representative strains for this QTL.
DNA synthesized from RNA uterine tissue was hybridized to Nimblegen mouse microarrays. Nineteen percent (3,436 transcripts) of the expressed uterine genes were modified in R3 uterus compared to those expressed in C57BL/6J. However, a significantly higher proportion of genes were deregulated when only the MMU1 spretus fragment or the two Led2 regions were considered.
Seven genes of the Led2a QTL were checked by quantitative RT-PCR. Focusing on genes of the Led2a interval and applying filters from bioinformatics databases, bibliography and the quantitative RT-PCR results, seven genes are proposed as putative actors of embryonic death: Trip12, Cab39, Psmd1, Ncl, Cops7b, Eif4e2 and Usp40.
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