Mouse (gene-engineered mice) ~Transgenic mice

Authors：

Category

Glycogene transgenic animals

Protocol Name

Authors

Furukawa, Koichi *
Department of Biochemistry II, Nagoya University Graduate School of Medicine

Ohmi, Yuhsuke
Department of Biochemistry II, Graduate School of Medicine, Nagoya University

*To whom correspondence should be addressed.

KeyWords

Transgenic

Reagents

●	cDNA clones which will be used as a transgene
●	An expression vector with an appropriate promoter to drive the expression of a inserted cDNA
●	Enzymes and competent cells (Takara Bio Inc., Otsu, Japan)

Instruments

●	Microinjector and microscope (Olympus, Tokyo, Japan)
●	Current apparatus for maintenance of experimental animals
●	Current apparatus and instruments to test behavioral abnormalities of mutant mice

Methods

Mouse (gene-engineered mice) ~Transgenic mice

1)	To construct a transgene (Fig. 1), cDNA of mouse β1, 4GalNAc-T (Takamiya K. et al. 1995) in pCDM8 is cleaved with Xho I, and the fragment containing the coding region is inserted into an EcoRI site of the plasmid pCAGGS (Niwa H. et al. 1991).	Comment 0

2)	The enzyme function of the transgene, pCAGGS/M2Tm is confirmed by the transfection into a mouse melanoma B16.	Comment 0

3)	The injection of the transgene into fertilized eggs was performed by the standard techniques, and Fo mice are obtained.	Comment 0

4)	F0 mice are mated each other, and tail DNAs of F1 mice are served for PCR screening to detect the β1,4GalNAc-T transgene. PCR amplification is performed with 0.1 μg of genomic DNA. PCR buffer (50 mM KCl/Tris, pH8.3/2.5 mM MgCl₂/0.01% gelatin), 0.1 μg each of primer (antisense primer sequence:440-457 in pTm3-5: 5’-AGAGCACTGATGTTGTACTC-3’/sense primer sequence:923-942: 5’-TTGACGCTGAGGAGCTGA-3’). 1 mM dNTP, and 0.6 unit TaqDNA polymerase (Wako Pure Chemical Industries Ltd., Osaka, Japan).	Comment 0

5)	Amplification is carried out by thermal cycler (Astec Co., Ltd., Fukuoka, Japan) for 3 min at 94°C and 30 cycles as follows: 1 min 94°C, 1 min 55°C, and 2 min 72°C.	Comment 0

6)	Analyses of the transgene expression by Northern blotting and/or RT-PCR. An example of Northern blotting is shown in Fig. 2.	Comment 0

7)	Direct detection of the protein products is also useful when antibodies are available.	Comment 0

Analyses of phenotypic changes in Tg mice are essentially same as described in the next chapter for gene knockout mice.

Comment 0

Discussion

If the transcripts from the transgene contain following properties, we can distinguish the transcripts from the transgene from that of the endogenous gene.

1. Different mRNA size

2. Different additional sequences in the transcripts

3. Tag-fused products for detection with tag-specific antibodies

Figure & Legends

Fig. 1. Construction of a transgene

Insert cDNA of mouse β1,4GalNAc-T in pCDM8 is cleaved with Xho I, and the fragment containing the coding region is inserted in a EcoRI site of the plasmid pCAGGS.

This figure was originally published in Glycobiology. 7(8):1111-20. 1997 "Genetic remodeling of gangliosides resulted in the enhanced reactions to the foreign substances in skin" Fukumoto S, Furukawa K. et al. Oxford University Press.

Fig. 2. Northern blotting of β1,4-GalNAc-T transgenic mice

mRNA expression levels of the gene were compared with those in the wild type mice.

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Date of registration：2014-02-26 17:52:42