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
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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