Acta Biotechnol. 13 (1993) 4, 373-383 Akademie Verlag

Characterization of Transgenic Mice Lineages

11. Transgenic Mice Expressing HBsAg Particles and Showing Female Sterility

BREZ ', A., CASTRO ', F. O., MART~NEZ ', R., FALCON', V., BARANOVSKY 2, N., BERLANGA 3, J., AGUIRRE ', A., INFANTE4, J., GUILLEN ', I., AGUILAR ', A., de la FUENTE ', J.

Mammalian Cell Genetics Division Centro de Ingenieria Genetica y Biotecnologia 31 Ave./158 and 190, Playa P.O. Box 6162 Havana, Cuba

Centro de Ingenieria Genetica y Biotecnologia 31 Ave./158 and 190, Playa P.O. Box 6162 Havana, Cuba

Centro de Ingenieria Genitica y Biotecnologia 31 Ave./158 and 190, Playa P.O. Box 6162 Havana, Cuba Department of Animal Models Instituto Finlay de Sueros y Vacunas Havana, Cuba

* Division of Chemical and Physical Analysis

' Clinical and Preclinical Trials Division

Summary

The tissue specificity of the expression of the hepatitis B surface antigen (HBsAg) in transgenic mice was studied. Northern blot hybridization and electron microscopy of various tissues revealed the higher HBsAg transcription levels confined to the liver and kidney, and to a lesser extent to the spleen and intestines. In electron microscopic studies we found HBsAg particles in the serum of transgenic animals to be positive by ELISA to HBsAg. The expression of the transgene was localized in siru in the ovary, liver, kidney, and spleen of transgenic mice, employing a polyclonal antiserum directed against the HBsAg and conjugated with protein A-gold. Surprisingly, FI transgenic females from the CB-I 04 line, expressing HBsAg in the serum, showed impaired fertility, although the ovarian function was not diminished. Normal off-spring were obtained after cross embryo transfer between transgenic (Tg) females to non-transgenic (Ntg) recipients or from Ntg embryos transferred to Tg recipients. We speculate that this phenomenon could be related to a disruption on a gene@) somehow involved in the reproductive performance of the transgenic females.

Introduction

Hepatitis B virus (HBV) infects humans and some other primates and may cause acute hepatitis or, in some patients, chronic hepatitis and hepatocellular carcinoma. Much is known about the structure, organization and replication of the HBV. The virus is a circular,

374 Acta Biotechnologica 13 (1993) 4

partially double-stranded DNA, 3.2 kb in length, which probably replicates via an RNA intermediate and has the capacity to be integrated randomly into the host genome. Acute hepatocellular injury generally occurs in the context of free, episomal, viral replication and is usually resolved by the elimination of virus-free and virus-infected cells. In the absence of viral clearance, viral sequences may be integrated into the human genome. The combined effects of a prolonged presence of integrated viral DNA with associated chronic hepatocellular injury and regeneration appear to predispose the infected hepatocyte to neoplastic transformation [ 11. Transgenic mouse systems have been widely employed as tools to analyze the biological effects of gene expression under physiological conditions that cannot be reproduced in cell cultures. The lack of susceptible tissue cultures for HBV propagation, as well as the narrow host of the virus, emphasize the value of the transgenic animals for elucidating HBV molecular biology. Previous studies have reported on the developmental regulation [2, 31, structural effects [l] and molecular pathogenesis of HBV [4, 5,6, 71. In this article, we present evidences of the tissue specificity in the expression of the hepati- tis B surface antigen (HBsAg) in a transgenic mice lineage. As determined by Northern blot analysis and immunoelectron microscopy, the higher HBsAg transcription level was detected in the liver and kidney, and to a lesser extent in the spleen and intestines. The results of the anatomopathological studies of organs from transgenic animals are also discussed in the paper with special emphasis on the accumulation of HBsAg particles in the ovary and on the female sterility observed in these animals.

Materials and Methods

Generation of Transgenic Mice

Transgenic mice lineage CB-104, CB-104-2 and CB-104-6 containing the HBV genome, except for the core sequence, were derived as previously described [8]. Transgenic animals were identified by the Southern blot analysis of the DNA obtained from tail segments [8,9, 101. All the animals used in this study were heterozygous for the transgene.

.Northern Blot Analysis

The total RNA was extracted from different tissues after homogenization in guanidium thyocianate, as previously described [ 111. For Northern blot analysis, 20 pg of the total RNA were electrophoresed in a 1.1% agaroxf2.2 M formaldehyde gel in 20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA. The RNA was then transferred to nitrocellulose filters (BA 85, SCHLEICHER & SCHUELL) and hybridized with (a-32P) dATP-labelled pUcHBV [8, 121. Hybridization was camed out overnight in 50% formamide, 0.1% SDS, 50mM NaH2P04, 10% dextrane sulfate, 5% DENHARDT'S solution, 5 x SSC (20 x SSC, 3 M NaCl, 0.3 M sodium citrate, pH 7) at 42 "C. After hybridization, the filters were washed twice in 1 x SSC/O.lYo SDS at room temperature and for 30 minutes a t 68 "C in 0.1 xSSC/O.l% SDS. The nitrocellulose filters were then exposed on RX films (FUJI) a t -70 "C for one week.

Characterization of Serum HBsAg Particles

Serum levels of HBsAg particles were determined by ELISA [8]. One ml of mice serum was centrifuged for 24 hours in a HITACHI SCP85H centrifuge at 50000 rpm at 4 "C. The pellet was used for electron microscopic analysis by the negative staining method [ 131.

Morphological Analysis

Tissue samples were fixed in 5% neutrally buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin, according to CHISARI et al. [4].

BREZ, A., CASTRO, F. 0. et al., Transgenic Mice Lineages I1 375

Immuno-Electron Microscopy

Tissue samples were fixed in 2.5% glutaraldehyde and were embedded in acrylic based embedding media (Lowicryl K4M, BALZERS). Thin sections were incubated with a drop of a specific rabbit polyclonal anti-HBsAg serum. Ultrastructural localization of immunoreactive sites was performed with a complex of protein A-colloidal gold according to KELLENBERGER et al. [14].

Results

Multiple Transcripts for the HBsAg are Present in Tissues of the CB-104 Founder Mouse

The plasmid pUcHBV contains the entire HBV genome except for 445 pb which correspond to the viral core sequences (Bgl I1 fragment, positions 1980-2425; [7]), inserted into the BamHI site of the plasmid pUC19 [8; Fig. 11. This plasmid directs the synthesis of HBsAg particles codified by a 2.1 kb mRNA transcribed from a promoter situated at position 3122 in the HBV genome [15].

A

EcoRI 2 .1 kb R t M Hind111

S I BannI/B9lII BanHI/BglII EcoRI

1900 bp

/ PUC19

B

5.4 kb -

1 2 3 4 5 6 7

Fig. 1 a. Schematic representation of the plasmid used for microinjection The whole viral genome (except for the core antigen) was inserted in the BamHI site of the pUC 19 plasmid. Fig. 1 b. Southern blot analysis of the integration of HBV sequences in mice The samples were digested with EcoRI restriction enzyme. Lines 1, 2, 3: 10 pg DNA of wild mouse (negative control). Line 4: 10 pg DNA from the transgenic mouse CB-104. Line 5 : molecular weight markers, DNA from lambda phage digested with Hind 111. Lines 6,7: 0,2 and 1 ng of pUC HBV Hind 111 plasmid.

376

1 2 3 4

- 5.4 kb

Acta Biotechnologica 13 (1993) 4

Fig. 2. Southern blot analysis of the progeny from CB-104 mouse 10 pg of each sample was digested with EcoRI restriction enzyme. Lines 1,2: non-transgenic animals. Lines 3, 4: transgenic animals.

As we previously reported [8], a hemizygotic line of transgenic mice was derived from the FO founder female CB-104. Southern blot analysis of DNA obtained from tail pieces of this female founder mouse CB-104 indicated the presence of multiple EcoRI fragments that hybridized with HBsAg gene sequences (Fig. 1). Additionally, the 2.0 kb EcoRI internal fragment was absent, which could indicate that the plJc19 EcoRI site had been lost during the integration process. Despite the fact that this observation has been reported before by others [16], we have obtained transgenic mice for this gene construction with all the EcoRi sites present in the integrated transgene sequences [8]. Northern blot analysis of specific HBsAg transcripts showed the existence of different expression patterns between the founder mouse CB-104 and the progeny (Fig. 2), in respect to the size of the transcripts, the tissue specificity and the relative mRNA levels of the HBsAg gene expression (Figs. 3,4). The founder mouse CB-104 showed high HBsAgmRNA levels in the liver and kidney, and lower mRNA levels in the spleen, intestines and muscles. However, the female progeny (CB-104 1-2 and CB-104 1-6) showed very low HBsAg mRNA levels only in the spleen (CB-104 I-2), intestines (CB-104 I-6), kidney (CB-104 I-6), muscles, and heart (CB-104 1-2) (Fig. 3). Furthermore, in the mouse CB-104, essentially three different sized HBsAg transcripts were found: 2.1 kb, 3.6 kb and 6.4kb. Of these transcripts, only the 2.1 kb mRNA corresponded to the transcription initiated by the viral promoter [I51 (Fig. 1). However, the most abundant and frequently transmitted to the progeny was the 3.6 kb mRNA (Fig. 4). The RNA isolated from the ovaries of transgenic animals was not enough to perform the Northern blot analysis.

The Accumulation of HBsAg Particles in the Ovary does not Cause an Apparent Tissular Damage

HBsAg particles present in the serum of transgenic mice were partially purified and characterized by immuno-electron microscopy employing a rabbit heteroserum. As shown in Fig. 5, filamentous and spherical particles with an average diameter of 22 nm were found, indistinguishable from particles found in the serum of HBV infected individuals

High amounts of HBsAg particles were found in the liver, kidney, spleen, and ovaries of the mouse CB-104 with no specific pattern of localization within the cell (Fig. 6). In the brain, lung, muscles, and heart, HBsAg particles were not detected. Histologically, in the

[6,7,171.

P~REZ, A., CASTRO, F. 0. et al., Transgenic Mice Lineages 11 377

Fig. 3. HBsAg mRNA levels in different organs from transgenic mice

liver of the CB-104 all neoplastic lesions were seen to occur against a background of ground glass cells, enlarged hepatocytes containing eosinophilic cytoplasmic and nuclear inclusions, focal hepatocellular necrosis, and reactive inflammatory infiltrate characterized by poly- morphonuclear leukocytes and macrophages. No abnormal histological feature was found in the other organs analyzed, including the ovaries.

1 2 3 4 5 6

Fig. 4. Northern blot of total RNA isolated from the transgenic mouse CB-104 and

Line 1 : RNA from E. Coli. Line 2 - 5: RNA from the spleen liver, intestine, kidney, respectively, of the CB-104 mouse. Line 6: RNA from the kidney of the CB-104 1-6 mouse.

CB-104 1-6

378 Acta Biotechnologica 13 (1993) 4

Fig. 5. Electron microphotography of HBsAg particles in the serum of transgenic mice

Reproductive Problems Found in the Transgenic Females are not a Result of a Failure in the Ovulatory Process Two litters were bred from the animal CB-104, by crossing with non-transgenic males, and approximately half of the F1 siblings were found to carry the transgene. Further attempts to get litters from the founder CB-104 as well as from the transgenic F1 females failed, and only a few fetuses were derived by cesaerian section (Tab. 1). Therefore, it has been impossible until now to generate a homozygotic transgenic animal, and the line has been kept “open”, through the crossing of transgenic males with non-transgenic females. As can be seen from the data in Tab. 1, reproductive problems were detected in all the females tested despite the level of expression of the HBsAg. For that reason, anatomo- pathological studies were conducted to identify possible structural damage responsible for this reproductive failure. However, no alterations were detected neither in the ovaries nor in the oviductal or uterine tissues (not shown). This finding correlates well with the existence of the ovulatory process in these animals. For this reason we designed an experiment in which embryos from transgenic females that were superovulated and matted to non-transgenic or to transgenic males were transferred to normal (non-transgenic) pseudopregnant females, while the embryos obtained from superovulated non-transgenic females were transferred to pseudopregnant recipient trans- genic females. As control groups for the standardization of the transfer procedure, normal embryos were transferred to normal females.

Tab. 1 . Score of reproductive failure in transgenic FO and F1 females

Females Detected Number of Implantations’ Fetusesb Live’ mated pregnant deliveries PUPS

7 7 0 26 16 3

a All implantation sites from all females, counted post morten after cesaerian section

‘ Live pups delivered by cesaerian section Full or non-full term fetuses still born at the moment of cesaerian section

P~REZ, A., CASTRO, F. 0. et al., Transgenic Mice Lineages I1 379

Fig. 6 . Immunocytochemical labelling of antibodies to HBsAg with a IgG-gold complex of Lowicryl K4M embedded tissues from the transgenic mouse CB-104 A - spleen ( x 12000); B - ovary ( ~ 2 0 0 0 0 ) ; C - kidney ( x 15000); D - liver ( x 30000); RER - rough endoplasmic reticulum; M - mitochondria; N - nucleus; L -lymphocyte

380 Acta Biotechnologica 13 (1993) 4

The transfer of transgenic embryos to non-transgenic uteruses resulted in a relative low pregnancy rate (56%) and more significantly in a very low efficiency of pups obtained from these embryos (Tab. 2). The control group for the standardization of the transfer procedure showed a normal pregnancy and normal efficiency rates for non-manipulated embryos according to the historical data of our laboratory (Tab. 2 ; [18]).

Tab. 2

Donor n Embryos Females Pups Efficiency" females transferred

transferred pregnant (n) ("/.I

8 9.87 2 6.66

Transgenics (T) 7 81 NT (9) 5 (56) Non-transgenics (NT) 3 30 T (3) 1 (33) NT 10 100 NT (10) 9 (90) 37 37.00

a - calculated as the number of pups/number of transferred embryos

Discussion

The founder female mouse (CB-104) employed in this report presented more than one integration site for the transgene sequences since multiple EcoRI fragments were hybridized in the Southern blot analysis of tail DNA (Fig. 1). The analysis.of individuals from the lineage showed that these bands were segregated independently as'would be expected for a Mendelian transmission (Fig. 2). The absence of the transgene internal EcoRI fragment in the Southern blot analysis of CB-104 DNA (Fig. 1) can be explained by the loss of the EcoRI site present in the pUC19 vector sequences. This suggests two possibilities for the different HBsAg transcripts found in the Northern blot analysis (Fig. 4): (1) differences observed in the expression of the HBsAg between the founder mouse and the progeny could be the result of differences in the transcriptional activity of transgenes integrated into different chromosomal locations, which segregate independently to the progeny, and (2) transcription is driven not only by the viral promoter, but also by promoters located 5' from the chromosomal integration site, and/or transcription ends in sites other than the viral polyadenylation site, which could be lost with the vector EcoRI site during the integration process. None of these possibilities can be definitively demonstrated. In the founder mouse CB-104, high HBsAg expression levels were found in the liver and the kidney (Fig. 4), which correlates well with previously reported findings [15,2, 191. Furthermore, the presence of the 2.1 kb HBsAg mRNA in most of the tissues studied (Fig. 4) suggests that the viral promoter is indeed active in cell types different from liver cells. This fact is in accordance with recent reports on the presence of receptors for HBV on some cells of extrahepatic origin, thus correlating with earlier observations indicating that hepatnaviruses are not strictly hepatotropic [20]. Several organ samples were taken for histological analysis. However, abnormal histological features were found only in the liver. The mechanism responsible for liver cell injury in human HBV infection is not clear. There is much speculation and considerable circum- stantial evidence, however, that injury is a result of a cytolytic immune response to HBV-encoded antigens. These antigens are expressed at the hepatocyte surface in association with polypeptides encoded by the major histocompatibility complex [4]. This is clearly not the case in our system, since the transgenic mice are immunologically tolerant to the HBV gene products. In the current transgenic system, liver cell injury is probably a result of the accumulation of non-secretable filamentous HBsAg particles within the endoplasmic

EREZ, A., CASTRO, F. 0. et al., Transgenic Mice Lineages I1 381

reticulum of the hepatocyte caused by the relative overproduction of the HBV large envelope polypeptide [4]. Initially, storage of this material causes ultrastructural and histological changes characteristic of the ground glass hepatocytes seen in the human HBV carrier state [21]. Eventually, however, toxic intracellular concentrations of HBsAg are reached, and the cells die [l]. It is possible that a similar process contributes to the pathogenesis of liver cell injury in HBV-induced liver disease in man. Since the production of the large envelope polypeptide appears to be tightly regulated during viral replication, such a process would imply dysregulation of HBV envelope gene expression, as could conceivably occur as a consequence of integration. There have been several reports on the strong relationship between chronic HBV infection and hepatocellular carcinoma [22,23,24,25]. However, very much is still unknown about the pathogenesis of the HBV infection. Recently, DUNSFORD etal. [26] showed that the overproduction of the HBsAg in transgenic mice initiated a process characterized by liver cell injury, inflammation and regenerative hyperplasia, which places large numbers of hepatocytes at risk in the development of transforming mutations, and which inexorably progresses to hepatocellular carcinoma. Thus, the development of hepatocellular carcinoma in transgenic mice overexpressing the HBsAg appears to occur due to the overproduction of HBsAg filaments. These filaments are accumulated within the endoplasmic reticulum of the hepatocyte and ultimately kill the cell [4]. By electron microscopy the transgene expression was detected in situ employing a polyclonal antiserum directed against the HBsAg and conjugated with protein A-colloidal gold. These results were in general in agreement to those found by the Northern blot analysis (Figs. 4 and 6). Surprisingly, a high level of expression was found in ovary tissues (Fig. 6), although no apparent tissular damage was detected. A reproductive failure was detected in the transgenic females that expressed the HBsAg in this lineage. This failure was manifested as a reduced frequency of litters obtained per female (in most cases no litters were obtained), and also as a high incidence of non-spontaneous delivery in the cases of the pregnant females. No reports on sterility of transgenic HBsAg-expressing females have been published ; however, impaired fertility of transgenic females has been documented for the human growth hormone (hGH) expressed in mice [27,28,29, 301. It has been clearly shown [28], that, in this case, luteal failure was a result of the unresponsiveness of corpora lutea to lactogenic hormons that led to the sterility of the females, although for the hGH transgenes there exists an additional problem related to the prolactin-like activity of the hGH. Human GH can overstimulate the tuberoinfundibular dopaminergic (TIDA) neurons which in turn interferred with the increase in prolactin release that normally follows matting and this leads to luteal failure. This does not seem to be the case for HBsAg transgenics decribed here since we have obtained normal pregnancy and deliveries from transgenic females which received non-transgenic embryos (Tab. 2). In our case, it is evident that the primary ovary response to the peptidic and steroid hormones of the reproductive cascade occurs, since both spontaneous and induced ovulation were achieved in transgenic females. Furthermore, viable embryos are indeed produced and can be cultured up to the blastocyst stage in vitro (data not shown). Whether the reproductive problem is inherent to the expression of the transgene by the growing embryo itself or it is a result of a physiological failure of the mother is still unknown. The lack of appropriate analytical systems for the detection of peptidic hormones as well as the reduced volume of blood that can be obtained from a mouse, make the establishment of the kinetics of the expression of peptidic hormones very difficult. In our experiments, the availability of recipient transgenic females for receiving transferred embryos was a limiting step. For this reason, the sample number for transgenic mothers as recipients of embryos was very low (n = 3), and therefore, it was difficult to withdraw any conclusion from this data. Further experiments will be needed to address whether transgenic

'

382 Acta Biotechnologica 13 (1993) 4

females will provide an adequate uterine environment for the development of normal embryos. Despite this fact, from the analysis of the data for transgenic embryos transferred to normal females, an important feature can be seen: Transgenic embryos might probably be somehow responsible for a decrease in the efficiency of pups that are delivered, since only a rough 10% of the transferred embryos gave birth to live fetuses while the normal rates for routine embryo transfer range between 25% and 40% [18]. Despite the fact that we can not definitely demonstrate the cause of the reproductive failure observed in the transgenic females of this lineage, we should take into account that these results correspond to a single lineage and, therefore, could reflect an individual genetic phenomenon. This phenomenon could be related to a gene(s) disruption that somehow affected the female reproductive process or could be responsible for an aberrate HBsAg gene expression that cause the observed failure. Finally, we could speculate that this phenomenon could be associated with the 5.4 kb EcoRI fragment found in the CB-104 founder female (Fig. 1) and in other individuals of the lineage (Fig. 2). This phenomenon may or may not also be associated with the 3.6 kb HBsAg transcript observed in the founder mouse and in the progeny (Fig. 4). This observation, even considered to be a particular genetic event resulting from a random transgene integration process, could permit the study of this specific locus with regard to the reproductive process in the mouse. More studies are currently in progress for a better understanding of the reproductive failure reported here.

Received 6 January 1993

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Book Review

P. GACESA, J. HUBBLE

Enzy mtechnologie

Berlin, Heidelberg, New York, London, Paris, Tokyo, Hong Kong, Barcelona, Budapest: Springer- Verlag, 1992 204 pp., 68 figures, 19 tables; DM 54,- ISBN 3-540-55183-2

The application of enzymes on an industrial scale was signicantly increased in the last decades especially by immobilization methods. The text-book on Enzyme Technology by P. GACESA and J. HUBBLE has been edited in order to inform the readers about the present state and to make them fit for a better understanding in this field of biotechnology. After a short introduction to the history and the significance of biocatalysis using enzymes, the matter of this book is divided into the following chapters: - Industrially used raw materials for enzyme production - Extraction and purification of enzymes - Kinetics of enzymatic reactions and reactor design - Enzymes in medicine and pharmaceutics - Influences of enzyme immobilization on stability and application - Enzymes in the agriculture and the food industry - Biosensors - Enzyme modification

Generally it could be said that the text-book represents the actual state of art. The matter is mediated in a very instructive and clear way. The many tables, graphs and schematic drawings are also favourable for easy understanding. It would be desirable, though, for the writers to include a more extensive handling of the possibilities of the production of microbial enzymes by discontinuous, fed-batch or continuous fermentation processes. The text-book will be useful, especially for students of microbiology, biochemistry, biotechnology, pharmaceutics, and the food sciences, and also for scientists and engineers working in these fields. G. KLAPPACH

- Outlook