**2. Nagase analbuminemic rats (NARs)**

Analbuminemic rats (Nagase analbuminemic rats, NARs) were first established from Sprague Dawley (SD) rats by Nagase et al. (1979). NARs show extraordinarily low serum albumin, hyperlipidemia and hormonal changes. However, their growth and reproduction rates do not differ from those of normal rats. The amount of total serum protein in NARs is similar to that seen in normal rats due to the increase in proteins other than albumin.

Analbuminemic Rat Model for Hepatocyte Transplantation 125

increase. B. Immunostaining for albumin in an F344 rat liver (a) and in young (b) and aged NAR livers (c). Albumin-positive hepatocytes are visible in the aged NAR liver (c) and are

In contrast to the hepatocytes of normal rats (Figure 1B a), hepatocytes of NARs are generally negative for immunohisotochemical staining of albumin (Figure 1B b). However, although NARs display extremely low serum albumin, albumin-positive hepatocytes sometimes can be seen in the liver tissue of NARs at low frequency. The number of such albumin-positive hepatocytes increases with aging (Figure 1B c) and after treatment with hepatic carcinogens (e.g. 3'-methyl-4-dimethylaminoazobenzene) (Makino et al., 1986). These albumin-positive hepatocytes are present as single or double cells in cross sections (Figure 1B c inset) and rarely form clusters consisting of more than three cells. These cells remain as single or double cells after liver regeneration following two thirds hepatectomy (PH), suggesting that albumin-positive hepatocytes may have a low proliferative capacity. Under these conditions, the prevalence of albumin mRNAs missing exons G and H and exons H and I increase along with exon H-skipped albumin mRNA (Figure 1A b). In addition, aberrant 60-kD albumin is generated in the liver (Kaneko et al., 1991). This abnormal albumin may be a translation product of mRNA that skips exons H and I, and may accumulate in the cytoplasm because of defects in extracellular albumin secretion,

Although NARs are derived from SD rats, the SD rats consist of out-bred strains. Therefore, SD rats may be genetically heterogeneous. We first tried to transplant SD rat hepatocytes into an NAR liver without immunosuppressants. However, this experiment was unsuccessful, most likely because the SD rats were immunogenetically heterogeneous. Cocultured spleen cells from NARs and SD rats displayed a higher rate of cell proliferation compared to cells in NAR/NAR or SD/SD cultures (Yokota & Ogawa, 1978). These results indicate that the immunological rejection may occur after the transplantation of the SD

In addition to the variety of abnormalities in serum proteins, lipids and hormones, NARs differ from SD rats in their susceptibility to tumorigenesis induced by chemical carcinogens in various organs. Notably, NARs are highly susceptible to urinary bladder tumors that are induced by *N*-butyl-*N*-(4-hydroxybutyl)nitrosamine (BBN) (Kakizoe et al., 1982) and less susceptible to hepatocarcinogenesis induced by diethylnitrosamine (DEN) and 2 acetylaminofluorene (2-AAF) (Asamoto et al., 1989). It is not clear whether the difference in the susceptibility to chemical carcinogens is the result of the analbuminemia in NARs or of differences in the genetic backgrounds of NARs and SD rats. To reduce the genetic variability, Takahashi et al. (Takahashi et al., 1988) established F344-alb rats with the genetic background of F344 rats. F344-alb rats are highly susceptible to BBN-induced urinary tumors compared to F344 rats (Takahashi et al., 1988) and are equally sensitive to DEN-2- AAF-induced hepatocarcinogenesis (Ohta et al., 1994). Because the only genetic difference between F344-alb rats and F344 rats is the aforementioned 7-base-pair deletion in the albumin gene in the F344-alb rats, the pairing of F344 and F344-alb rats can be used for cell

usually present as single cells (inset) or two-cell clusters.

resulting in positive albumin immunostaining (Kaneko et al., 1991).

**3. F344-congenic analbuminemic rats (F344-alb rats)** 

transplantation without using immunosuppressants.

hepatocytes into the NAR liver.

Analbuminemia in NARs is an autosomal recessive trait. Therefore, the serum albumin levels are nearly normal in the F1 hybrids of NARs and normal rats.

The molecular basis for analbuminemia in NARs is a deletion of base 5 to base 11 at the 5' end of the 9th intron of the albumin gene (Figure 1A a) (Esumi et al., 1983). Most of the albumin mRNA in NARs shows a precise deletion of exon H, which is skipped during albumin pre-mRNA processing following the deletion in the 9th intron (Figure 1A b) (Shalaby & Shafritz, 1990). The deletion of exon H causes a frameshift in the mRNA and the occurrence of a translation termination signal at the 7th codon of exon I, preventing production of the albumin protein (Shalaby & Shafritz, 1990).

Fig. 1. A. The structures of the albumin gene and mRNA in the Nagase analbuminemic rat (NAR). a. Seven base pairs (the 5th to 11th base pairs from the start of the intron) are deleted between exons H and I (9th intron). b. Exon H is skipped in most albumin mRNAs during albumin pre-mRNA processing (1). With aging and treatment with hepatocarcinogens, the levels of albumin mRNA transcripts that skip exons H and I (2) and exons G and H (3)

Analbuminemia in NARs is an autosomal recessive trait. Therefore, the serum albumin

The molecular basis for analbuminemia in NARs is a deletion of base 5 to base 11 at the 5' end of the 9th intron of the albumin gene (Figure 1A a) (Esumi et al., 1983). Most of the albumin mRNA in NARs shows a precise deletion of exon H, which is skipped during albumin pre-mRNA processing following the deletion in the 9th intron (Figure 1A b) (Shalaby & Shafritz, 1990). The deletion of exon H causes a frameshift in the mRNA and the occurrence of a translation termination signal at the 7th codon of exon I, preventing

Fig. 1. A. The structures of the albumin gene and mRNA in the Nagase analbuminemic rat (NAR). a. Seven base pairs (the 5th to 11th base pairs from the start of the intron) are deleted between exons H and I (9th intron). b. Exon H is skipped in most albumin mRNAs during albumin pre-mRNA processing (1). With aging and treatment with hepatocarcinogens, the levels of albumin mRNA transcripts that skip exons H and I (2) and exons G and H (3)

**NAR (young) NAR (aged)** 

levels are nearly normal in the F1 hybrids of NARs and normal rats.

production of the albumin protein (Shalaby & Shafritz, 1990).

increase. B. Immunostaining for albumin in an F344 rat liver (a) and in young (b) and aged NAR livers (c). Albumin-positive hepatocytes are visible in the aged NAR liver (c) and are usually present as single cells (inset) or two-cell clusters.

In contrast to the hepatocytes of normal rats (Figure 1B a), hepatocytes of NARs are generally negative for immunohisotochemical staining of albumin (Figure 1B b). However, although NARs display extremely low serum albumin, albumin-positive hepatocytes sometimes can be seen in the liver tissue of NARs at low frequency. The number of such albumin-positive hepatocytes increases with aging (Figure 1B c) and after treatment with hepatic carcinogens (e.g. 3'-methyl-4-dimethylaminoazobenzene) (Makino et al., 1986). These albumin-positive hepatocytes are present as single or double cells in cross sections (Figure 1B c inset) and rarely form clusters consisting of more than three cells. These cells remain as single or double cells after liver regeneration following two thirds hepatectomy (PH), suggesting that albumin-positive hepatocytes may have a low proliferative capacity. Under these conditions, the prevalence of albumin mRNAs missing exons G and H and exons H and I increase along with exon H-skipped albumin mRNA (Figure 1A b). In addition, aberrant 60-kD albumin is generated in the liver (Kaneko et al., 1991). This abnormal albumin may be a translation product of mRNA that skips exons H and I, and may accumulate in the cytoplasm because of defects in extracellular albumin secretion, resulting in positive albumin immunostaining (Kaneko et al., 1991).
