1. 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).

2. 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)

3. 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.