导航：首页-->生物技术-->分子生物学技术指导（在线分子生物学技术方法)

• DNA Preparation
• Gene Transfer
• Embryo Transfer
• Transgenic Identificatioin
• Others

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• Transgenic Outline (University of Michigan Transgenic Animal Model Core)
  This is an outline of the steps necessary to obtain a transgenic mice. Simply put, the investigator constructs a transgene with a promoter and a structural gene (for example a reporter gene such as lacZ or a transcription factor). DNA is prepared and microinjected into fertilized mouse eggs. Potentially transgenic mice are born. Transgenic founders are identified and bred to produce offspring for analysis  
    
• Transgenic animals (Brinkmann)
  Two techniques are introduced: Microinjection of linear DNA into the pronucleus and Transfer of Embryonic Stem Cells into Blastocysts

DNA Preparation

• Preparation of DNA for Microinjection (Pharmacological Sciences Consortium Core)
  Restrict DNA, gel purification, ethanol precipitation... 
    
• Sample preparation for microinjection (Brinkmann)
  Microinjection is the loading or transfer of a dissolved substance into a living cell. The microscopic tip of the glass microcapillary has an inner diameter between 0.2 and 1 µm [1]. This capillary is back loaded with the substance to be transferred into the cells cultured for microinjection. Typical substances include purified antibodies, DNA, RNA, peptides, or oligonucleotides.
     
• DNA Purification for Transgene Microinjection (U. of Virginia Transgenic Mouse Core Facility)
  
• DNA Purification for Microinjection (University of Michigan
  Transgenic Animal Model Core)
    
• DNA Ultrapurification for Microinjection (Gary A. J. Brown)
  Several methods are described in detail.
    
• DNA Preparation for microinjection or electroporation (Transgenic and ES Cell Services at OSU)
    
• BAC DNA Purification for Microinjection (University of Michigan
  Transgenic Animal Model Core)
  This method is used to purify BAC DNA for microinjection into fertilized mouse eggs. This method yields clean, non-sheared DNA
     
• DNA Purification for Electroporation (University of Michigan
  Transgenic Animal Model Core)
     
• Preparation of Transgene DNA for Microinjection (New England Medical Center)

Gene Transfer

• Electroporation of ES Cells and Isolation of H/R Clones  (Bowtell Lab)
   
• Electroporation of ES Cell (Nagy Lab)
       
• ES / MEF Cell Culture and Electroporation of Targeting construct (Gary Brown)
    
• DNA Pronuclear Injections (Transgenic and ES Cell Services at OSU)
    
• Microinjection of Single Cells in Culture (Mitshison Lab)

Embryo Transfer

• Blastocyst Transfer (Bowtell Lab)
  
• Blastocyst Transfer (PMCI)
    
• Blastocyst Embryo Transfer into Pseudopregnant Recipient Female Mouse (Gary Brown)
  Detailed protocol with illustration 

Transgenic Identification

• Genotyping Transgenic Mice by PCR (University of Michigan Transgenic Animal Model Core)
  Test mice for the presence of the transgene by PCR. It is provided for those investigators who are unfamiliar with mouse genotyping and who need to establish a genotyping method.
     
• Yolk Sac DNA Preparationor Southern or PCR  (U. of Virginia Transgenic Mouse Core Facility)
     
• DNA Purification for PCR only from yolk sacs, toes, or 2 mm Tail (U. of Virginia Transgenic Mouse Core Facility)
      
• Mouse Tail Digestion Procedure (Pharmacological Sciences Consortium Core)
    
• PCR of DNA from Ear Clip Tissue (Transgenic and ES Cell Services at OSU)
    
• Genomic Southerns of DNA from Tail Clips (Transgenic and ES Cell Services at OSU)
    
• Isolation of DNA from Mouse Tail Biopsies (University of Michigan Transgenic Animal Model Core)
    
• Genomic DNA preparation (Pfaff Lab)
  Prepare genomic DNA from ES cell or tail for genotyping. 
     
• Non-organic Isolation of Tail DNA (Jackson Labs)
  Using tail tissue directly for PCR after proteinase K digestion
    
• DNA Extraction from Tail Biopsies (Jackson Labs)
  Phenol:chloroform extraction method
     
• Mouse Tail (or organ) Biopsy DNA Extraction (The Cancer Center at Washington University)
    
• Isolation of DNA from Mouse Tail Biopsies (University of Michigan Transgenic Animal Model Core)
    
• DNA preparation from mouse tails (Dr. Ornitz's Lab, Washington University Medical School) 
  Avoid Shearing of the DNA by limiting mixing 
  to inversion and gentle shaking. Vortexing of samples is not required to perform the following protocols. Avoid contamination by rigorously cleaning pipetman and using autoclaved tips during manipulation of genomic DNA and reagents to be used with genomic DNA...
     
• Preparation of Mouse Tail DNA for Dot Blots or PCR  (Eric Mercer)
  These procedures were originally devised in Richard Palmiter's lab for use with tail dots (DNA spotted onto a nitrocellulose filter and probed for a transgene; quicker than doing southerns), and subsequently modified for PCR preps.
    
• Preparation of Copy Standards for PCR Genotyping (University of Michigan Transgenic Animal Model Core)
  The purpose of spiking tail DNA with a known amount of transgene DNA is to produce copy standards. These are used to verify the sensitivty of your PCR genotyping assay and to establish transgene copy number in Southern blot analysis.
    
• PCR Analysis of Tail DNA (University of Michigan
  Transgenic Animal Model Core)
     
• LacZ Staining of Embryos (Pfaff Lab)
      
• LacZ Cell Staining and β-galactosidase Embryo Staining (Dr. Ornitz's Lab, Washington University Medical School) 
  Protocols for cell and embryo staining.
    
• AP Assay and FGF Binding (Dr. Ornitz's Lab, Washington University Medical School) 
  FGFR-AP-FGF Binding Assay, PCR Assays for Knockout Mice, RNase Protection Assay:The bound receptor was quantified by measuring the initial rate of substrate hydrolysis at 405 nm and same amount of proteins was used in the binding assays...
      
• Whole Mount LacZ Staining (Nagy Lab)
    
• Whole Mount Human Placental Alkaline Phosphatase Staining (Nagy Lab)
    
• Whole Mount Immunoflurescentstaining (Nagy Lab)
    
• Whole Mount RNA in situ Hybridization (Nagy Lab)
  RNA probe labeling and purification, embryo preparation and more...
    
• Immunocytochemistry Staining of Embryos (Pfaff Lab)
    
• X-gal & AP Staining Protocol (Cepko/Tabin Lab)
  Very detailed protocol with background information and several applications.
    
• RNA Whole Mount In Situ Hybridization (Cepko/Tabin Lab)
  protocol for both mouse and chick embryos

• Timeline for Transgenic Mouse Production/Analysis (University of Michigan Transgenic Animal Model Core)
    
• Transgenic Strain Staining Assay (Jackson Labs)
  Lac-Z Detection in Tail Biopsies
      
• Mating Transgenic Mice (University of Michigan Transgenic Animal Model Core
   

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