Chul-Sang Lee

Incidence of hepatocellular carcinoma in transgenic mice expressing the hepatitis B virus X-protein

BACKGROUND/AIMS Chronic infection with hepatitis B virus is a high-risk factor for hepatocellular carcinoma in humans. The HBV X-protein, a multi-functional viral regulator, has been suspected to play a positive role in hepatocarcinogenesis, as demonstrated by the high incidence of hepatocellular carcinoma in HBx-expressing transgenic mice, although it is still controversial. The aim of this study was to generate transgenic mice expressing the HBV X-gene under authentic promoter control and to test whether the gene products can cause hepatic tumors. METHODS Three transgenic mouse lines were generated by microinjecting the X-gene construct into hybrid (C57BL/6 x DBA) eggs. Gene expression was tested by protein and mRNA analyses. During an observation period of 18 months, mice were sacrificed and organs subjected to histologic examinations. RESULTS Grossly defined hepatocellular carcinomas reproducibly were observed in mice expressing the X-protein, which were investigated through six generations from the age of 11 to 18 months. Among 14 transgenic mice investigated from the age of 11 to 18 months, 12 were found to have hepatocellular carcinoma, grossly or microscopically. The lesion of the hepatocellular carcinoma disclosed a significant increase in the proliferating cell nuclear antigen in the nuclei. CONCLUSION The incidence of hepatocellular carcinoma (86%) in our HBV X transgenic mice may be highly significant, since, except for one case, HBV X-gene transgenic mice produced in other laboratories did not develop liver tumor or any other pathologic phenomena.

Production of biologically active human granulocyte colony stimulating factor in the milk of transgenic goat

We have developed a transgenic female goat harboring goat beta-casein promoter/human granulocyte colony stimulating factor (G-CSF) fusion gene by microinjection into fertilized one-cell goat zygotes. Human G-CSF was produced at levels of up to 50 μ g/ml in transgenic goat milk. Its biological activity was equivalent to recombinant human G-CSF expressed from Chinese hamster ovary (CHO) cell when assayed using in vitro HL-60 cell proliferation. Human G-CSF from transgenic goat milk increased the total number of white blood cells in C57BL/6N mice with leucopenia induced by cyclophosphamide (CPA). The secreted human G-CSF was glycosylated although the degree of O-glycosylation was lower compared to CHO cell-derived human G-CSF.

Embryo recovery and transfer for the production of transgenic goats from Korean native strain, Capra hircus aegagrus.

During the breeding season in Korea (September 1997 to April 1998), a goat embryo recovery and transfer program using a Korean native strain (Capra hircus aegagrus) was performed for the production of transgenic goats. Donors were synchronized with norgestomet implants and superovulated by a combined treatment with FSH and hCG. The treatment regime induced a consistent and efficient superovulation rate of 90% from donors with an ovulation rate of 12.1+/-0.5. 50.5% of the recovered oocytes/embryos were fertilized and most of them were at the 1-cell stage. After DNA microinjection, a total of 188 embryos were immediately transferred to naturally cycling or hormonally synchronized recipients with two or three embryos per animal. There was a tendency for the pregnancy rate of naturally cycling recipients to be higher (38.9%) than that of hormonally synchronized recipients (25.7%). When the recipients were classified into a two or three embryo-received group, the embryo viability was markedly decreased from 58.3% in two embryo-received group to 35.3% in three embryo-received group, without an increase in the kidding rate. This resulted from a high occurrence of abortions or stillbirths from the multiple-pregnant recipients which had received three embryos. This indicated that the transfer of two embryos per recipient is highly recommendable for an optimal embryo survival in Korean goats. Altogether, 188 embryos were transferred to 71 recipients and two transgenic Korean goats were produced from 25 offspring.

Efficient Integration of Short Interspersed Element-flanked Foreign DNA via Homologous Recombination

We investigated whether mouse short interspersed elements (SINEs) could influence the recombination frequency of foreign DNA. Vectors harboring a reporter gene in combinations of SINEs B1 and/or B2 or a portion of long interspersed element-1 were prepared and tested in vitro by a colony assay using HC11 murine mammary epithelial cells and in vivo by microinjection into fetilized mouse eggs. In transfected HC11 cells, the number of colonies surviving G418 selection increased by 3.5-fold compared with control when the reporter was flanked by fused B1-B2 sequences. Similar results were obtained from microinjection study; in fetuses 11.5 days post coitum, transgene positives in control and SINE-flanked vectors were 16 and 53%, respectively. Individual B1- and B2-harboring vectors showed equivalent activities with each other, as determined by the colony assay (2.8-fold versus 3.2-fold compared with control). We determined the contribution of homologous recombination to the SINE-mediated increase in integration frequency through a polymerase chain reaction-based strategy; in more than half of embryos transgenes underwent homologous recombinations involving B1 sequences. These results demonstrate that the SINE sequences can increase the integration rate of foreign DNA and that such an increase is most likely due to the enhancement of homologous recombination.

Potent and stage-specific action of glutathione on the development of goat early embryos in vitro.

The effect of glutathione (GSH) addition on the development of 1‐ or 2‐cell goat early embryos in vitro was examined. Embryos were collected from superovulated Korean black goat (Capra hircus aegagrus) and cultured for 6 days in synthetic oviduct fluid medium supplemented with either bovine serum albumin (BSA) or serum. Without GSH addition, almost all embryos could not develop beyond 8‐ to 16‐cell block. However, GSH addition greatly improved in vitro development of early embryos to blastocyst stage, and its action was highly dependent on the presence and source of proteins supplemented into the culture medium. Among the protein‐supplemented cultures, GSH effect was most prominent in 10% FBS‐supplemented culture, in which the proportion (91%) of blastocysts developed from early embryos was much higher than that of BSA‐ (42–64% depending on its content) or goat serum (GS)‐supplemented cultures (21%), or even than that of somatic cell‐supported co‐culture (60%). As well as in terms of the morphological development, mean cell number of blastocysts (185 ± 12) developed from FBS condition was significantly higher than that of blastocysts developed from any other culture conditions and moreover comparable to that of blastocysts developed in vivo (190 ± 9). The viability of these blastocysts was finally confirmed by their term development (6/12) from embryo transfer. To delineate action time of GSH during embryo development, GSH was treated at 1‐day intervals through 6‐days culture periods excepting the last day. In the GSH‐treated embryos at day 3 of culture, which corresponds to the time of in vitro 8‐ to 16‐cell block stage, the proportion of blastocyst was markedly increased up to 77% of cultured embryos and conversely that of the arrested embryos was decreased to 7%. In the embryos treated later, however, their developmental potency decreased abruptly. Therefore, these results clearly demonstrated that GSH could greatly improve the in vitro development of goat early embryos by specifically acting on the 8‐ to 16‐cell block stage during in vitro development, suggesting that GSH may be one of the important regulators on the development of goat embryos in vivo. Mol. Reprod. Dev. 57:48–54, 2000.

An efficient expression of Human Growth Hormone (hGH) in the milk of transgenic mice using rat {beta}-casein/hGH fusion genes

In order to produce human growth hormone (hGH) in the milk of transgenic mice, two expression vectors for hGH differing in their 3′ flanking sequences were constructed by placing the genomic sequences of hGH gene under the control of the rat Β-casein gene promotor. The 3′ flanking sequences of the expression constructs were derived from either the hGH gene (pBCN1GH) or the rat Β-casein gene (pBCN2GH). Transgenic lines bearing pBCN1GH expressed hGH more efficiently than those bearing pBCN2GH in the milk (19-5500 Μg/mL vs 0.7-2 Μg/mL). In particular, one of the BCN1GH lines expressed hGH as much as 5500 ±620 Μg/mL. Northern blot analysis showed that the transgene expression was specifically confined to the mammary gland and developmentally regulated like the endogenous mouse Β-casein gene in the mammary gland. However, a low level of nonmammary expression was also detected with more sensitive assay methods. In conclusion, the rat Β-casein/hGH fusion gene could direct an efficient production of hGH in a highly tissue-and stage-specific manner in the transgenic mice and the 3′ flanking sequences of hGH gene had an important role for the efficient expression.

A hybrid bovine beta-casein/bGH gene directs transgene expression to the lung and mammary gland of transgenic mice.

We investigated spatial and temporal expression of bGH controlled by two different sizes (1.8 kb and 15 kb) of 5′-flanking sequences of the bovine ß-casein in transgenic mice. In the 1.8-kb promoter-containing mice, bGH expression was specifically confined to lung and mammary gland at lactation. While mammary gland expression was highly variable depending on the lines, lung expression was relatively constant with a high level in most lines. Moreover, this dual-tissue specificity of bGH expression was consistently retained in all of the 15 kb-promoter-containing mice, although a low ectopic expression was sometimes detected in salivary gland or brain. During mammary gland development in the 1.8-kb promoter-containing mice was mammary gland expression first detected at lactation, following the bovine rather than murine pattern of ß-casein expression. In contrast, lung expression was almost constant regardless of mammary gland developmental state or sex. Therefore, it can be concluded that a combination of the bovine ß-casein promoter and bGH gene directs a distinct dual-tissue specific bGH expression with different regulatory mechanisms between mammary gland and lung and as little as 1.8-kb promoter is sufficient for the proper regulation of the bovine ß-casein gene in mammary gland.

Improved development of DNA-injected bovine embryos co-cultured with mouse embryonic fibroblast cells

The in vitro development of DNA-injected bovine zygotes, produced in vitro, was compared when cultured with or without mouse embryonic fibroblasts (MEF). The in vivo viability of the embryos produced in these in vitro culture systems was assessed by single or double transfer to recipients taken to term. For these experiments, in vitro fertilized oocytes were not injected (Experiment 1) or were injected with pBL1 gene (Experiment 2) and then cultured for 2 days in CR1aa medium supplemented with 3 mg/ml BSA at 38.5 degrees C in a humidified atmosphere of 5% CO(2) in air. Embryos that developed to the 4- to 8-cell stage at the end of this period were randomly assigned to the two cultured systems and cultured for a further 5 days in groups of 10 to 15 embryos in 0.75 ml medium. These two culture systems were CR1aa medium alone or co-culture with MEF in CR1aa medium supplemented with 10% fetal bovine serum (FBS). Every 48 h, 0.5 ml of the medium was replaced with fresh CR1aa medium and at Day 5 of culture, both media were supplemented by the addition of 5.56 mM glucose and 1x GMS-X supplement solutions. Results were assessed as morphological development of the embryos and data were analyzed by Chi-square test or Students t-test. The development rate of in vitro fertilization (IVF)-derived embryos co-cultured with MEF (24.4%, 49/201) was significantly higher than those cultured alone (14.4%, 28/194; P<0.05) in Experiment 1. There was a similar difference between the treatments in the proportions of embryos which reached the hatching stage or hatched (10.9%, 22/201 vs. 4.1%, 8/194, respectively; P<0.05). DNA-injected embryos co-cultured with MEF (13.7%, 28/205) showed a higher developmental rate than that of the embryos cultured without MEF (6.7%, 13/193; P<0.05) in Experiment 2. Following the transfer to recipients of one or two DNA-injected blastocysts, the pregnancy rates for two culture systems were similar (MEF co-culture 27.4%, 23/84; CR1aa culture 24. 2%, 16/66). However, the numbers of calves born alive from these pregnancies were higher on the MEF co-culture group (82.6%, 19/23) than the CR1aa culture group (56.2%, 9/16). It was concluded that in vitro embryo development to the blastocyst stage and subsequent in vivo development to term of DNA-injected bovine embryos was improved in comparison to culture in CR1aa alone when the last 5 days of in vitro culture were in a MEF co-culture system.

Effect of short interspersed element sequences on the integration and expression of a reporter gene in the preimplantation‐stage mouse embryos

Based on the assumption that foreign DNA sequences may have increased chance of integration into the host genome if they are flanked by high copy‐numbered genomic sequences such as SINEs (short interspersed elements), we investigated the integration frequency of Lac Z reporter gene flanked by a fused B1/B2 in an in vivo system using pronuclear microinjection technique in the mouse. The SINE‐flanked DNA showed a 4‐fold increased integration frequency of the reporter gene than the control DNA (63% vs. 16%). Moreover, the level of β‐galactosidase expression, estimated from the X‐Gal staining intensity in transgenic embryos, was greatly higher in SINE‐carrying DNA. These results suggest that the SINE sequences can serve a very useful tool in improving the efficiency of current transgenic animal technology. Mol. Reprod. Dev. 56:366–371, 2000.

Sequence and functional analysis of an upstream regulatory region of human HOXA7 gene

The Hox genes have been known to be involved in pattern formation during vertebrate development through differential expression along the anteroposterior body axis. Human homologue of position-specific regulatory region of murine Hoxa-7 was cloned from human genomic library. The restriction map of the 18-kb insert was determined, of which a 3.9-kb region was sequenced. Homology plot between the murine and the corresponding human sequence showed high sequence conservation over 70% in several regions. The homologous region has been reduced to about 1.1 kb (HCR: human control region), which contained several putative factor binding sites. The function of HCR was analyzed in transgenic mice and turned out to be a position-specific regulatory element of human, setting the precise anterior boundary of expression in transgenic embryos; at day 12.5 post-coitum a distinct anterior limit of expression was noted at the level of C5 in neural tube and spinal ganglia in transgenic embryos. These results indicate that the regulatory sequences as well as the molecular mechanism for Hox gene expression are highly conserved among vertebrates.