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REPORTER KNOCK-IN MOUSE
BY KALYANI RAJALINGHAM
In order to create a reporter knock-in mouse, one must first create a knock-in construct, and
introduce them into ES cells via methods like electroporation. The target vector construct
would include a 5’-arm, a 3’-arm, a LacZ gene, a neomycin gene, and HSV-tk (Figure 5).
Homologous recombination between the target vector, and the wild-type allele (in genome)
should in theory incorporate the foreign DNA segment of interest. Subsequently, the ES cells
must be screened using a negative selection method – antibiotics like neomycin – and
cultivate the cell culture. Genotyping via Southern Blotting can be carried out as a
confirmation procedure (Iwatsuki et al. 2010).
Generated Foxk2+/LacZ
ES cells are then injected into the host blastocyst which can then be
implanted into a female foster mouse or surrogate mother. The foster mouse will give birth to
chimeric mice. Chimeric mice are crossed with normal mice to obtain Foxk2+/LacZ
mice;
heterozygotes can then be crossed to generate homozygous Foxk2LacZ/LacZ
mice. The tip of the
tail is then cut off, and used for genotyping.
Analysis of Reporter gene expression is carried out via X-gal staining. In most cases, three
samples are strained: Foxk2+/LacZ
, Foxk2LacZ/LacZ
, and the wild-type (control). Histological
slices or sections of the organism can be photographed.
Fluorescent immunohistochemistry using antibodies against Foxk2, and β-galactosidase can
further be used to validate the results obtained from the X-gal staining of the three strains
(Foxk2+/LacZ
, Foxk2LacZ/LacZ
, and the wild-type) (Iwatsuki et al. 2010).
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Figure 5: Target vector construct (LacZ = gene for production of β-galactosidase; Neo =
Neomycin (negative selection method); HSV-tk = lethal marker (positive selection
marker); 5’Arm = region homologous to genomic DNA at 5’ end; 3’Arm = region
homologous to genomic DNA at 3’ end)
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REFERENCES
Iwatsuki, Ken, Masatoshi Nomura, Atsushi Shibata, Reiko Ichikawa, Patricio L.m. Enciso,
Lixiang Wang, Ryoichi Takayanagi, Kunio Torii, and Hisayuki Uneyama. "Generation and
Characterization of T1R2-LacZ Knock-in Mouse." Biochemical and Biophysical Research
Communications 402.3 (2010): 495-99. Web.