Báo cáo khoa học: Development of an HSV-tk transgenic mouse model for study of liver damage

Development of an HSV-tk Transgenic Mouse Model for Study of Liver Damage

Authors: Yan Zhang, Shu-Zhen Huang, Shu Wang, Yi-Tao Zeng

Field: Genetics, Medical Science

Document Content: This study describes the development and characterization of a novel transgenic mouse model engineered to study liver damage. The model utilizes the herpes simplex virus thymidine kinase (HSV-tk) gene, regulated by an albumin promoter/enhancer, enabling liver-specific expression. Upon administration of ganciclovir (GCV), a prodrug, the HSV-tk enzyme converts it into a toxic metabolite, leading to selective depletion of hepatocytes and inducing liver injury. This inducible system allows for controlled induction of liver damage, offering a valuable tool for investigating the mechanisms of liver diseases and potential therapeutic strategies. The research details the construction of the HSV-tk vector, generation of transgenic mice, confirmation of transgene integration and expression, and the subsequent induction and analysis of liver damage through histological, biochemical, and molecular methods. The study also addresses potential side effects, such as male infertility observed in some transgenic lines due to ectopic HSV-tk expression in the testis.

Detailed Table of Contents:

• Development of an HSV-tk transgenic mouse model
• Keywords
• Correspondence
• (Received 13 January 2005, revised 23 February 2005, accepted 7 March 2005)
• doi:10.1111/j.1742-4658.2005.04644.x
• The herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system that selectively depletes cells expressing HSV-tk upon treatment with GCV has provided a valuable tool for developing a new animal model expressing the desired tissue damage.
• In this paper, an HSV-tk vector with an albumin promoter/enhancer was constructed.
• Based on the favourable killing effect on Hep-G2 cells by the recombinant construct, the HSV-tk transgenic mouse strains were developed.
• One strain of the TK transgenic mouse (TK5) was studied intensively.
• Integration of the target gene was confirmed primarily by PCR.
• Fluorescence in situ hybridization following G-banding analysis demonstrated that the insertion site was located at 2F1-G3.
• The hepatocyte-specific transcription and expression of HSV-tk was verified by reverse transcription (RT)-PCR as well as by immunohistochemical staining.
• When two second-generation mice (TK5-F1 and TK5-F2) were injected with GCV, the pathogenic alterations in the liver were readily identified, including the appearance of vaculation in the hepatocytes with inflammatory infiltration in the liver, and diffuse proliferation of hepatocytes.
• In addition, the blood test demonstrates a significant increase of serum alanine aminotransferase, aspartate aminotransferase and total bilirubin.
• In conclusion, the transgenic mouse model with hepatocyte-specific expressed HSV-tk developed hepatitis with administration of GCV, had morphological and clinical chemical characteristics indicative of hepato-cellular disease and should be useful for the study of inducible liver-specific diseases.
• The morbidity of severe liver disease is usually very high, seriously threatening the patient’s health.
• Availability of animal models expressing related hepatic disor-ders should provide a means of studying the pathogenic mechanism of such diseases.
• Among these are the genetically engineered animal models.
• Unfortunately, currently available transgenic models are unsatisfactory for experimental use, because those transgenic mice expressing toxic protein often die too early due to the over-expression of toxic protein in such a vital organ.
• Others, for example the alb-uPA transgenic mouse and FAH knockout mouse developed in the 1990s can be main-tained only with constant medical treatment [1,2].
• In 1989, Heyman et al. [3] developed a new trans-genic mouse in which ablation of a specific cell type is TK-dependent.
• In such a transgenic mouse, the inserted herpes simplex virus thymidine kinase (HSV-tk) gene products can phosphorylate certain nucleoside analogues such as ganciclovir (GCV) that are not metabolized by conventional cellular enzymes.
• Phosphorylated nucleoside analogues such as GCV triphos-phate are potent toxic metabolites for cells.
• Nevertheless, neither GCV nor the HSV-tk alone is harmful to cells.
• Hence, this conditional cell-depleting effect is achieved by expressing HSV-tk with a cell-specific promoter.
• It has been used for depletion of lymphoid cells, growth hormone-secreting cells, inter-leukin-2 and interleukin-4-expressing cells, dendritic cells or fibroblasts under the control of a cell-specific promoter [4-9].
• Such a system is used in the transgenic rats of Kawasaki et al. [10], in which the rats develop experimental hepatitis on administration of GCV.
• The genome of the mouse is much better characterized than that of the rat and the cost of producing and maintain-ing transgenic mice is less than for rats.
• The high conservation and strong liver-specific regulatory machinery of the mouse serum albumin cluster makes it appropriate for use as a promoter for hepatic-speci-fic expression [11,12].
• In this study, HSV-tk transgenic mice were produced, in which the inserted gene is regulated by an albumin enhancer/promoter; liver injury is readily induced in this model.
• Among five founder transgenic mice generated, only one (TK5♀) transmitted the transgene to progeny through the germ line by mating with male -/- wild-type KM mice.
• Therefore, the F1 and F2 generations of TK5 were used for the inducible hepatic injury.
• In addition, the founder TK3♂ was also used for preliminary ana-lysis of the relationship between expression level and histopathological changes.
• Results
• Liver damage in transfected Hep-G2 cells after treatment with GCV
• The PCMV-TK vectors were transfected into Hep-G2 cells, which were then induced with GCV.
• The transfected cells started to detach on third day after a single exposure to 40 µmol·L¯¹ GCV.
• Hep-G2 cells transfected with pLLTK started to detach on day 5, and maximal expression was achieved on day 7 after GCV treatment.
• Cell apoptosis was recognized in both of the two groups mentioned above; sick or damaged cells were seen to swell and burst.
• By contrast, the control cells transfected with vector pcDNA 3.1/zeo(+) grew and proliferated normally.
• The Hep-G2 transfected pLLTK showed completely different morphology as compared to the control cells (Fig. 1A).
• Such increased cell death could also be assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide assay.
• The survival rate of Hep-G2 cells was reduced significantly after trans-fection with HSV-tk (Fig. 1B).
• Detection of inserted transgene and monitoring of the pedigree of mouse family TK5 by PCR
• A total of 182 eggs were microinjected and subse-quently reimplanted into eight pseudopregnant foster mothers, of which five became pregnant and gave birth to 36 mice.
• Among them, six mice showed the inser-tion of the HSV-tk gene as detected by PCR.
• The integration rate was 16.7% (6/36).
• One line of trans-genic mice is female; the transgene is transmitted to the offspring at a rate of about 50% according to Mendel’s laws (Fig. 2A).
• Chromosomal localization of transgene integration as demonstrated by fluorescence in situ hybridization (FISH)
• More than 50 metaphases were analysed for each transgenic mouse.
• All of the metaphase cells showed one positive hybrid signal.
• According to the standard idiograms of mouse chromosomes, the integration site is located at 2F1-G3 in TK5-F1-455 (Fig. 2B).
• The integration site of TK5-F2-327 was similar to that of TK5-F1-455 (data not shown).
• Reverse transcription (RT)–PCR of tissue-specific expression of HSV-tk in transgenic mice
• RT-PCR showed that the 390 bp specific band of HSV-tk was detectable only in the transfected cells, liver and testicle.
• It was not detectable in the cells used as negative control, or in blood, kidney, pancreas, intestine, brain, skin or heart, even though an internal control band of 190 bp ẞ-actin was present in all of the samples (Fig. 3).
• HSV-tk protein expression in the liver of transgenic mice
• Immunohistochemical staining was performed using a polyclonal rabbit-(anti-HSV-tk) Ig.
• The yellowish-brown staining sites were located mainly in the nucleus of hepatocytes integrated by the HSV-tk gene, and the HSV-tk-positive cells were distributed scattered or clustered in liver lobules, located mainly around the central vein, and occasionally in the periportal areas (Fig. 4A), while there was no staining in the liver of the wild-type mice (Fig. 4B).
• Simultaneously, the positive signal can be observed in both the nucleus and the cytoplasm after GCV treatment (Fig. 4C).
• The staining cells account for 20-30% of the total hepatocytes of TK5-F1-455 (Fig. 4A), whereas in mouse TK3, there were approximately 60-70% HSV-tk staining hepato-cytes, with visible slight yellowish-brown signals in the focal necrosis, but several regenerative foci (regener-ating parenchyma hepatocytes) displayed reduced or no staining (Fig. 4D).
• The percentage of HSV-tk-positive hepatocytes in the F2 mice (TK5-F2-327) of TK5 was similar to those of TK5-F1-455 (data not shown).
• Hematoxylin and eosin staining for histological analysis
• Microscopic analysis of the livers of GCV-treated HSV-tk mice (F1 and F2) showed that the diseased liv-ers display a number of abnormalities, including the appearance of apoptosis bodies, hepatocyte vaculation, lymphocyte infiltration, hepatocyte megalocytosis, and diffused proliferation of hepatocytes (Fig. 5A).
• In transgenic mouse TK3, mutifocal coagulation necrosis was evident in the liver (Fig. 5B).
• Histological analysis of the kidney showed no apparent abnormity in the GCV-treated transgenic mice and wild-type mice (data not shown).
• Biochemical analysis of the blood
• Twenty-one days after the injection of GCV, the values of alanine aminotransferase (ALT), aspartate amino-transferase (AST) and total bilirubin were significantly increased in the TK5-F1 transgenic mice (P < 0.05), whereas there were no significant increase in the wild-type control mice.
• The value of creatinine was not altered significantly in either group (Fig. 6).
• The changes of the four serum values in the F2 generation of the TK5 family and mouse TK3 showed similar results (data not shown).
• Discussion
• We have generated a number of transgenic mice for liver damage, in which the HSV-tk gene was regulated by an albumin promoter/enhancer.
• The excellence of this mouse model is that liver damage and its extent in HSV-tk mice can be induced and controlled by GCV treatment.
• When the mice are injected with GCV, the pathologic changes and biochemical abnormalities, including vaculation of the hepatocytes, inflammatory infiltration, diffuse proliferation of hepatocytes as well as a significant increase of serum ALT, AST and total bilirubin, can be easily recognized.
• However, renal function is not affected by GCV treatment.
• This indicates that GCV at the dosage used in this study is associated with toxin-mediated hepatocyte damage in our HSV-tk mice.
• We used FISH and RT-PCR to investigate trans-gene integration and HSV-tk expression in various tissues of the transgenic mice.
• FISH indicated that the transgene was stably integrated in the genomes of the mouse family (TK5), and the expression of HSV-tk was readily detectd in the liver and the testis of TK5 family, but was not detectable in other tissues, such as blood, kidney, pancreas, intestines, brain, skin and heart.
• It indicated that the recombinant construct dri-ven by the albumin/enhancer that we used in this study was highly tissue specific.
• Immunohistochemical analysis confirmed that the HSV-tk protein was expressed specifically in the liver.
• In TK3 mice, approximately 60-70% of the total hepatocytes showed HSV-tk expression, and 20-30% of the liver cells in the TK5 family gave positive results.
• The discrepancies of levels of HSV-tk expression between these two mouse strains may be due to differences in HSV-tk gene integration sites, that may be caused by random integration of the transgene.
• Recent studies showed that HSV-tk converts the nontoxic prodrug GCV into GCV-triphosphate, which can cause chain termination and single-strand breakage upon incorporation into DNA.
• Although blocking of DNA synthesis of GCV is especially toxic for dividing cells, it can also cause damage of nondividing cells, such as hepatocytes, and liver toxicity of HSV-tk [13,14].
• This provides the basis for selected hepatocyte killing using a hepatocyte-specific promoter in vivo.
• Hepatocyte replication was not a prerequisite for this effect, indicating that interference with DNA syn-thesis during S phase of the cell cycle is not the only mechanism of toxicity of phosphorylated GCV.
• Furthermore, although the exact mechanism by which suicide genes kill the HSV-tk-expressing cells is not yet clear, apoptosis has been considered to be a major contributor to GCV killing [15-18].
• Song et al. [19] have shown that GCV induced HSV-tk expressing cells into apoptosis, thus inhibiting the growth of ovarian cancer cells.
• Shibata et al. [20] injected the HSV-tk vec-tor into rats with bladder cancer and observed apopto-sis of bladder cancer cells.
• Kawasaki et al. [10] created an AL-HSV-tk transgenic rat that expressed HSV-tk in hepatocytes, in which apoptosis was demonstrated after treatment with GCV.
• The administration of GCV elicited leukocyte infiltration and induced chronic hepatitis [21,22].
• Although in the hepatitis model the precise role of Kupffer cells is unclear, it is possible that they are involved in inflammation [10].
• Activated Kupffer cells release cytokines and chemokines that activate and transport T cells [23-25].
• It seems that hepatitis in the rat is primed by hepatocyte apoptosis [10].
• To examine the immunological mechanisms involved in cell killing using the HSV-tk/GCV system, HSV-tk-transduced human hepatocellular carcinoma (HCC) cells were implanted subcutaneously into im-munocompetent syngeneic mice.
• After GCV treatment, marked infiltration by lymphocytes including CD4+ and CD8+ T cells, apoptosis of cells was induced, and significant reduction or even complete regression of tumours was achieved.
• Conversely, no significant inhibitory effects on tumour formation were observed in athymic nude mice.
• The results indicate that T cell-mediated immune responses may be a critical factor for achieving successful cell killing using the HSV-tk/GCV system [26].
• Administration of GCV to mice and rats injected with adenovirus encoding HSV-tk caused extensive signs of liver degradation with negli-gible survival rate [14].
• Microscopic analysis of the GCV-treated HSV-tk rat model of Kawasaki et al. [10] revealed moderate hepatocyte vacuolation and an increased number of inflammatory cells.
• In this study, we found that there was more severe focal necrosis of the liver tissue in TK3 than in TK5 mice.
• Moreover, the liver regenerating focus was more evident in TK3, in which clones of transgene expression-deficient cells were formed as detected by immunohistochemistry.
• The reasons for the different pathological changes between these two mouse strains are not clear.
• We suggest that these differences may be associated with the quantities of HSV-tk expressing cells.
• In addition, the patchy focal necrosis and regeneration in the TK3 liver could be explained by the possibility that this founder mouse may be a mosaic as approximately 5-10% of founder mice showed mosaicism of some sort (either multiple integration sites or patchy cellular distribu-tion).
• Boucher et al. [27] compared the efficacy of the HSV-tk/GCV system in two human carcinoma cell lines after exposure to GCV and found that the killing effect depended on the concentration of the tk enzyme, the number of cells expressing HSV-tk, different cell types and the overall confluence of the HSV-tk expres-sing-cells.
• These results emphasise the importance of cell-specific metabolism in HSV-tk/GCV-mediated cytotoxicity.
• In conclusion, the killing of cells with HSV-tk/GCV is a complex interactive sequence of biochemical and cellular events involving incorporation and accumulation of the monophosphorylate deriv-ative of GCV into DNA, disruption and inhibition of the cell cycle, gap junction metabolite transfer, and apoptosis.
• Thus, we conclude that the exact mecha-nisms may differ in: (a) different cell types; (b) differ-ent species; (c) the concentration of GCV used; (d) the quantity of cells expressing HSV-tk; and (e) the distri-bution of cells expressing HSV-tk.
• It is of interest that male HSV-