**4. Hepatocyte transplantation using F344 and F344-alb rats**

To monitor the transplantability of hepatocytes in the liver, transplanted cells are detected using markers that are specifically expressed in the donor cells. For this purpose, various immunohistochemical and enzymatic histochemical procedures are used to visualize the proteins that are specifically expressed in the donor hepatocytes, such as *E. coli* galactosidase (-gal) (ROSA26-transgenic mice) (Mao et al., 1999) or green fluorescence protein (GFP-transgenic mice) in the normal mouse liver (Chiocchetti et al., 1997), dipeptidyl peptidase IV (DPPIV) in the DPPIV-/- rat liver (Gupta et al., 1995) and fumarylacetoacetate hydrolase (FAH) protein in the FAH-/- mouse liver (Hamman et al., 2005). When combining cells from a male donor and a female recipient, Y chromosome-specific *in situ* hybridization techniques are also useful (Eckert et al., 1995).

In the F344/F344-alb model, albumin-positive donor F344 hepatocytes in the F344-alb liver are detected using immunohistochemical staining of albumin and PCR-based techniques detecting normal albumin mRNA and genomic DNA. In addition, the functionality of F344 hepatocytes can be repeatedly evaluated by taking blood samples and examining the elevation of serum albumin levels.

In immunohistochemical staining for albumin, the technique used for tissue fixation is important because albumin tends to diffuse out of hepatocytes during tissue fixation. We usually fix the liver tissues by perfusing periodate-lysine-paraformaldehyde (PLP) fixative through the portal vein. We then incubate the sliced hepatic tissues in the same fixative overnight at 4℃ and then embed the tissues in paraffin (Ogawa et al., 1993). These procedures preserve the antigenicity of albumin and the morphological integrity of hepatic tissues. Antibodies that specifically recognize rat albumin are commercially available.

Donor F344 hepatocytes can also be detected by RT-PCR targeting normal albumin mRNA (Ohta et al., 1993a). Because albumin mRNA in F344-alb rats lacks exon H, RT-PCR with primers targeting exons G and I amplify both the normal and the short albumin mRNA from RNA that is isolated from recipient livers. In addition, primers targeting exons H and I specifically amplify normal albumin mRNA. Because the expression levels of exon Hskipped albumin mRNA in the F344-alb liver are low but relatively constant, the small amounts of normal albumin mRNA in the recipient F344-alb rat livers can be quantified using abnormal albumin mRNA as an internal standard (Ohta et al., 1993a).

The donor-derived F344 hepatocytes can also be detected by the amplification of the albumin genomic DNA sequences using DNA isolated from the recipient livers (Ogawa et al., 1993). Because of the 7-base-pair deletion in the 9th intron of the analbuminemic albumin gene, PCR amplification of amplicons spanning these sequences can differentially detect normal and abnormal genes, which can be used to quantify the expression of the normal albumin gene, using the abnormal albumin gene as an internal control (Ogawa et al., 1993). This method is applicable to hepatocytes and other cell types, such as bone marrow cells (Arikura et al., 2004; Inagaki et al., 2011).

The functionality of the transplanted F344 hepatocytes is evaluated based on the increased serum albumin levels in the recipients. Because the serum albumin level in F344-alb rats is extremely low, a small increase in albumin is detectable by sensitive methods such as the enzyme-linked immunosorbent assay (ELISA). However, conventional gel-electrophoretic

To monitor the transplantability of hepatocytes in the liver, transplanted cells are detected using markers that are specifically expressed in the donor cells. For this purpose, various immunohistochemical and enzymatic histochemical procedures are used to visualize the proteins that are specifically expressed in the donor hepatocytes, such as *E. coli* galactosidase (-gal) (ROSA26-transgenic mice) (Mao et al., 1999) or green fluorescence protein (GFP-transgenic mice) in the normal mouse liver (Chiocchetti et al., 1997), dipeptidyl peptidase IV (DPPIV) in the DPPIV-/- rat liver (Gupta et al., 1995) and fumarylacetoacetate hydrolase (FAH) protein in the FAH-/- mouse liver (Hamman et al., 2005). When combining cells from a male donor and a female recipient, Y chromosome-specific *in situ* hybridization

In the F344/F344-alb model, albumin-positive donor F344 hepatocytes in the F344-alb liver are detected using immunohistochemical staining of albumin and PCR-based techniques detecting normal albumin mRNA and genomic DNA. In addition, the functionality of F344 hepatocytes can be repeatedly evaluated by taking blood samples and examining the

In immunohistochemical staining for albumin, the technique used for tissue fixation is important because albumin tends to diffuse out of hepatocytes during tissue fixation. We usually fix the liver tissues by perfusing periodate-lysine-paraformaldehyde (PLP) fixative through the portal vein. We then incubate the sliced hepatic tissues in the same fixative overnight at 4℃ and then embed the tissues in paraffin (Ogawa et al., 1993). These procedures preserve the antigenicity of albumin and the morphological integrity of hepatic tissues. Antibodies that specifically recognize rat albumin are commercially available.

Donor F344 hepatocytes can also be detected by RT-PCR targeting normal albumin mRNA (Ohta et al., 1993a). Because albumin mRNA in F344-alb rats lacks exon H, RT-PCR with primers targeting exons G and I amplify both the normal and the short albumin mRNA from RNA that is isolated from recipient livers. In addition, primers targeting exons H and I specifically amplify normal albumin mRNA. Because the expression levels of exon Hskipped albumin mRNA in the F344-alb liver are low but relatively constant, the small amounts of normal albumin mRNA in the recipient F344-alb rat livers can be quantified

The donor-derived F344 hepatocytes can also be detected by the amplification of the albumin genomic DNA sequences using DNA isolated from the recipient livers (Ogawa et al., 1993). Because of the 7-base-pair deletion in the 9th intron of the analbuminemic albumin gene, PCR amplification of amplicons spanning these sequences can differentially detect normal and abnormal genes, which can be used to quantify the expression of the normal albumin gene, using the abnormal albumin gene as an internal control (Ogawa et al., 1993). This method is applicable to hepatocytes and other cell types, such as bone marrow cells

The functionality of the transplanted F344 hepatocytes is evaluated based on the increased serum albumin levels in the recipients. Because the serum albumin level in F344-alb rats is extremely low, a small increase in albumin is detectable by sensitive methods such as the enzyme-linked immunosorbent assay (ELISA). However, conventional gel-electrophoretic

using abnormal albumin mRNA as an internal standard (Ohta et al., 1993a).

**4. Hepatocyte transplantation using F344 and F344-alb rats** 

techniques are also useful (Eckert et al., 1995).

elevation of serum albumin levels.

(Arikura et al., 2004; Inagaki et al., 2011).

assays are unsuitable because the serum of F344-alb rats contains proteins with molecular weights similar to that of albumin, and the levels of other proteins may increase to compensate for the lack of albumin (Ohta et al., 1993b). Therefore, conventional gel electrophoretic assays may falsely detect protein levels that are much higher than those of the actual albumin levels.
