Gender Of The Whole Genome Amplification In The Early Mouse Embryo Single Blastomere Diagnosis

Objective : To construct an animal model about x-linked disease with PCR technique for following human preimplantation genetic diagnosis, a procedure involving embryo biopsy and genetic analysis, which was performed at mouse embryos in the 4- or 8-cell-stage.Methods 1.To select health female KM mice between 4 to 6 weeks, 10 IU PMSG were injected into abdominal cavity of every mouse , equal doses HCG were repeated 48 hours later. Equal amounts male mice between 6 to 8weeks were allocated into single cage in the ratio of 1:1 pair-matching to above female mice. The pregnant mice were picked out while the plugs were founded in their vagina in the next morning .The pregnant mouse was killed 39-42 hours later, excising mouse whole oviducts and partial uterus ,the mouse embryos in 4- or 8-cell-stage were collected into M16 medium by flushing the tubes through infundibulum with M2 medium under dissecting microscopy. The percentage of grades of embryos were recorded. These embryos continue to develop in M16 at 37℃ at CO₂ hatching cabinet and be inspected periodedly. The dilateral spermaductus were ligatured in the other mature male mice .Confirming infertility ,they were pair-matched to female mice undergoed superovulation similar to above procedure. Females which had visible positive vaginal plug next morning were consider as pseudopregnant mice .2 . A series microneedle for holding, aspirating and resolving were made up from 1mm×90mm glass pipette with special instruments. Embryos were divided randomly into three groups, a control and two experiments. Experiment embryos were divided into conventional and simplified groups according to different zona drilling by Tyrode's acid with micromanipulation. Data were compared between two experiment groups as follow biopsy time, percentage of intact embryos and intact single blastomeres. The cleavage 8 hr later of the three groups embryos were observed,and the rate of development of morula .3. Single blastomeres were transferred into 0.5ml eppendorf tubes containing 5μl double dilution water. Pre-amplification of entire genomes of single cells were performed by the methods of primer extension pre-amplification (PEP) after digestion with protease K. The primer of ZFX/ZFY gene was designed on primer 3 software(http://www.mit.edu) by scanning mouse gene bank withBLAST(http://www.ncbi.nlm.nih.gov/BLAST). The ZFX/ZFY gene was...