Screening And Identification Of The Differentially Expressed Genes In The Abnormal Limb Of Mouse Embryo Following Exposure To MNNG

Congenital malformation is one of the major causes in infant death. Our knowledge about the cause and mechanisms through which these defects are manifested is limited. Current estimated about 20% of all birth defects are due to genetic factors and 10% are due to environmental factors. The advent of combinatorial chemistry and computer-aid drug design has led to a recent upsurge in the number of chemicals. Their potential teratogenecity cannot be ignored. Understanding the molecular control of abnormal development can provide enormous groundwork for detection and extrapolation among species by identifying candidate genes and some known genes involved in the congenital malformation bad been identified. With the establishment of a series of molecular strategy, a detailed understanding of the genome-wide gene expression of abnormal development is becoming a reality. The vertebrate limb is a powerful model system for studying the cellular and molecular interaction that determine morphological pattern during embryonic development. MNNG is a known teratogen that can induced limb malformation. In this study, we used MNNG as test material to induce ICR mice embryo limb malformation. The result of experiment show that a spectrum of malformation involving the palate, ribs and limbs is produced. Limb defects are prominent with exposure on day 12, When the type of limb malformation are examined, an unusual pattern was observed. Limbs on the left side were more frequently malformed than limb on the right side and bindlimbs were more frequently malformed than forelimbs. The phenotypes of affected limb include oligodactyly, polydactyly and truncated limb mainly. Skeletal preparation show defect of metacarpals. The loss of tibia and fibia is account for the truncated limbs. Left-right asymmetry of ribs defect has been observed especially. To identify altered gene expression in abnormal and normal developmental limbs of mice GD 18 embryo, we employed a suppression subtractive hybridization(SSH) technique to generate two subtracted cDNA libraries. A set of eDNA clones include 172 SSH inserts amplified by PCR was screened, and 25 clones were shown by dotblotting to be differentially expressed in the malformed limb. 12 of 25 clones were set to be sequenced analysis and 10 significant clones were aligned in GeneBank. One clone with increased expression were identical to mus musculus peroxisomal biogenesis factor 3 (Pex3) mRNA and the others contain limited sequence homology to the previously reported mus musculus or 8-12 days embryo mus musculus expressed sequence tag. This work thus provide a starting point for understanding a molecular profile of abnormal, limbs development and chemical teratogenic mechanism that could ultimately provide an essential aid to developing strategies or the prevention, early detection of birth defect.