转基因

互联网2013-09-06

1705

DNA Preparation
Gene Transfer
Embryo Transfer
Transgenic Identificatioin
Others

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

DNA Preparation

Preparation of DNA for Microinjection (The University of Illinois at Chicago Cancer Center)
The vector sequences will have to be cut out from the transgene construct to release the insert (gene fragment) that you wish to have injected. This fragment must have a promoter, a start codon, a cDNA/genomic DNA/or reporter gene that you want to express, and a stop codon with a poly A region.
Preparation of DNA for Microinjection (Pharmacological Sciences Consortium Core)
Restrict DNA, gel purification, ethanol precipitation...
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)

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

Transgenic Screening (The University of Illinois at Chicago Cancer Center)
Two methods (southern and PCR) are discussed
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.
DNA Isolation from Murine Tails without Phenol Chloroform (Immunology Resource)
Detailed protocol and recipes
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)
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)
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

Others

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