Genomic mapping within the albino-deletion complex using individual early postimplantation mouse embryos

Abstract

Sensitive methods for analysis of DNA from limited amounts of tissue are often difficult, error prone, and time consuming. Here, we describe a procedure for molecular analysis of individual early postimplantation mouse embryos by Southern analysis. The procedure involves embedding single embryos in agarose before lysing and deproteinizing in situ. The embedded DNA can be digested with restriction enzymes and analyzed by standard Southern-blotting procedures. The procedure is sensitive enough to detect single-copy sequences in embryos as early as day 6.5 of development. We have used the technique to genotype embryos homozygous for an embryonic lethal deletion. Normally, the lethal phenotype associated with such mutations is identified by a retrospective statistical analysis of abnormal embryos produced from a heterozygous cross as compared to those produced from a control cross. Now, if associated with a detectable DNA abnormality, the mutant embryo can be genotyped directly. We also report the use of this method for mapping cloned markers relative to deletion breakpoints. This approach can save considerable time since mapping would conventionally be done using restriction fragment length polymorphisms (RFLPs) detected in Mus musculus/Mus spretus interspecies hybrids. Using this procedure, we have been able to redefine the distal limits of the region of Chromosome (Chr) 7 containing a gene (eed) needed for development of the embryonic ectoderm.

N-Ethyl-N-nitrosourea-induced prenatally lethal mutations define at least two complementation groups within the embryonic ectoderm development (eed) locus in mouse Chromosome 7

“…Recently, embryos homozygous for the c e~ deletion, a member of the original Di group of c deletions (Russell et al 1982), were found to exhibit a phenotype identical to that found in embryos homozygous for c zzDsD (Sharan et al 1992). c 2~ does not extend as far as D7CwrllD, the locus defined by the distal breakpoint of the c re)so deletion (Sharan et al 1992).…”

“…Recently, embryos homozygous for the c e~ deletion, a member of the original Di group of c deletions (Russell et al 1982), were found to exhibit a phenotype identical to that found in embryos homozygous for c zzDsD (Sharan et al 1992). c 2~ does not extend as far as D7CwrllD, the locus defined by the distal breakpoint of the c re)so deletion (Sharan et al 1992). Mice homozygous for either the c TM or c zz2K deletions, which both delete the segment from Fah to Mod-2, progress completely through development but die shortly after birth (Gluecksohn-Waelsch 1979;Russell et al 1982).…”

“…This neonatal lethality is probably due, at least in part, to a deficiency of fumarylacetoacetate hydrolase (Klebig et al 1992;Ruppert et al 1992). Thus, all available genetic mapping data indicate that at least a segment of the earlier-acting, postimplantation-lethal eed gene(s) must lie between Mod-2 and the distal breakpoint of the c 2~ deletion (Sharan et al 1992; Fig. 1).…”

“…Correspondence to: E.M. Rinchik (Niswander et al 1988(Niswander et al , 1989Sharan et al 1991Sharan et al , 1992. The eed phenotype was originally characterized by the failure of embryos homozygous for the c 11DsD deletion to develop beyond the early stages of gastrulation.…”

“…The bracket above l(7)5Rn and l(7)6Rn indicates that these two loci cannot yet be ordered with respect to each other. The shaded box represents the embryonic ectoderm development (eed) interval, which is currently defined as the region of non-overlap between the distal breakpoints of the c 3/~ (or c H2K) and c z~ deletions (Sharan et al 1992). See Rinchik and associates (1992) for locus references.…”