reporter knock-in

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).

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)

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.