GENERATION OF TRANSGENIC MICE
A laboratory manual
Lluís Montoliu (1993)
Updated version (1995)
Additional notes (1997)

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CONTENTS of this MANUAL  (116 printes pages) 

Oocyte Injection: Generation of Transgenic Mice

1.- ESTABLISHMENT OF MICE COLONIES

    1.a Normal males
    1.b Young females
    1.c Vasectomized males
    1.d Pseudopregnant females
    1.e Wet-nurse females (foster mothers)

2.- OOCYTE INJECTIONS

    2.a Superovulating females, weekly plan
    Weekly Plan
    2.b Collecting oocytes
    2.c Microinjecting oocytes
        2.c.1 Preparation of DNA for oocyte microinjection
        2.c.2 Microinjection of DNA into the pronucleus of the oocyte
            2.c.2.a Making holding pipettes
            2.c.2.b Making microinjecting needles
            2.c.2.c Microinjecting DNA into the eggs
     2.d Culturing oocytes
     2.e Transferring oocytes to recipient pseudopregnant  females

3.- ANALYSIS OF THE OFFSPRING

    3.a Pregnancy ratios. Littersizes
    3.b Weaning of the pups
    3.c Identification of individual mice (earmarks)
    3.d Tail biopsies
    3.e Isolation of high-molecular weight genomic DNA from mouse tails
    3.f Identification of transgenic mice
        3.f.1 Digestion of genomic DNA with restriction enzymes
        3.f.2 Southern analysis. Blotting onto nylon membranes
            3.f.2.a Radiolabelled DNA-size markers
        3.f.3 Slot-blot analysis of genomic DNA
        3.f.4 Synthesis of radiolabelly labelled probes
            3.f.4.a RNA probes (riboprobes)
            3.f.4.b DNA probes (random priming)
        3.f.5 Hybridization
        3.f.6 Oligonucleotide labelling and hybridization
        3.f.7 PCR analysis
    3.g Analysis of expression of the transgene
        3.g.1 Isolation of RNA. Urea/LiCl method
        3.g.2 Isolation of RNA. Guanidine thiocyanate method
        3.g.3 Checking the quality of the RNA samples
        3.g.4 Northern blot analysis
        3.g.5 RNase protection method
    3.h Use of reporter genes: LacZ (bacterial ß-galactosidase)
        3.h.1 Quantification of ß-galactosidase activity in extracts
        3.h.2 Detection of LacZ activity on cryostat sections
        3.h.3 Detection of ß-galactosidase activity by direct staining of samples
    3.i Interpretation of the results. Examples and conclusion.

4.- CRYOPRESERVATION OF MOUSE EMBRYOS

5.- MICROINJECTION OF YEAST ARTIFICIAL CHROMOSOMES

Bibliography



 

HOW TO RECEIVE A COPY OF THIS MANUAL? 

This manual for the generation and analysis of transgenic mice is freely available to all members of the Scientific Community that would be interested. This manual is intended only for academic, non-profitable and non-commercial purposes. It contains a collection of first-hand laboratory protocols originally derived from the laboratory of Prof. Günther Schütz (DKFZ, Heidelberg, Germany) that have been subsequently updated and/or slightly modified. In order to receive an electronic version of this manual (PDF file) please follow these indications:

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Last update: 26 March 2004