Department of Biological Sciences

Stem cell biology, germ cells, gene targeting, meiosis, spermatogenesis, transgenic fish

Research interests:

During early 1980sin the lab of Prof Xu Xianju at Wuhan university,I analyzed karyotypes in more than 30 fish species, developed procedures for short-term primary fish cell cultures, DNA-replication banding patterns in cyprinids, for sister chromatid differential staining (SCD) and chromosome preparations from single developing fish embryos. These procedures were applied to analyze cell cycle parameters, to monitor mutagens and to examine the ploidy levels of induced gynogens and polyploids. I developed a heat-shock procedure and succeeded in inducing allo- and auto-tetraploidy at high efficiency of up to 48% in cyprinids. These procedures of chromosome preparation are standards which were taught in EMBO workshops and are currently cited. I described a novel type of sex determination in one Clupeiform fish, Colia brachygnathus which is ZW-ZO.

In Prof Schartl's lab, Wuerzburg, I isolated the growth hormone (GH) gene from the silver carp (Hong & Schartl, 1992) and two metallothionein (MT) genes from the rainbow trout (Hong & Schartl, 1993). Both promoters together with the human counterpart were analyzed for expression regulation in response to heavy metal ions by CAT reporter assay in 6 fish cell lines and one human cell line as well as developing fish embryos ((Winkler et al., 1992). The two MT promoters and the carp ß-actin promoter were linked to the silver carp GH gene as well as the seabream GH cDNA, resulting in a first generation of all-fish transgene constructs for inducible and constitutive expression (Carvari et al., 1994). The constructs were transfected into EPC cells from common carp and total RNA was prepared. By Northern analysis I demonstrated that the GH transgene were transcribed in response to metal induction and mRNA of the predicted size of 1.2 kb was detected. I undertook RT-PCR to clone the transgene-derived cDNA. Sequencing confirmed a perfect match between the cloned and predicted cDNA sequences. These results demonstrated for the first time the efficient, inducible transcription of a homologous transgene and correct mRNA processing in fish cells. These constructs were successfully used for producing transgenic carps.

Embryonic stem (ES) cells are now well known to public and represent one of the most active research fields. ES hold enormous potential for basic study on early vertebrate development, for genetic engineering and for cancer biology and most importantly, cell therapy by transplantation.

ES cells were first established in the mouse in 1981. Since then tremendous endeavor has been attempted in many other mammalian species without any success until 1996, which disappointed the scientific community to work on ES cell derivation from other important organisms including human. It was thought that the ability to derive ES cells might be limited only to the mouse. One of major challenges of ES derivation is to inhibit their spontaneous differentiation. In mice this is achieved by using feeder layer cells and/or leukemia inhibitory factor. With Prof Schartl (Wuerzburg), I developed a feeder-free system to culture medaka blastula-derived embryonic cells (Hong & Schartl, 1996), succeeded in the establishment of several diploid medaka ES (MES) cell lines that share many common features with mouse ES cells (Hong et al., 1996, 2004a, b). Most importantly, I demonstrated that MES1, one of the MES cell lines, was chimera-competent (Hong et al., 1998). This series of my work demonstrated that the ability to derive ES cells does also exist in other vertebrate, possibly also in human. In fact, human ES cells were reported 2 year later in 1998.

Although ES cells as a universal source of differentiated cells hold promise for cell therapy, a major challenge is how to obtain a pure population of differentiated cells. To this issue I made use of the medaka ES cells and established a first model of directed differentiation in fish, where introduced expression of melanocyte-inducing transcription factor as a cell lineage master regulator is sufficient for driving ES cell differentiation along the pigment cell lineage (Bejar et al., 2003).

Medakafish model for gene targeting. ES cells are widely used for the production of knockout mice for analyzing genes' functions and for generating animal models of human diseases. Knockout production relies on gene targeting through homologous recombination in ES cells and germline transmission of ES cells in chimeric animals. This ES cell technology has largely restricted again to the mouse. Medaka is an excellent model for establishing and fully exploiting the ES cell technology, because of its daily egg production, easy, external and transparent embryology, and most importantly, a unique vertebrate in which ES cell lines have been made available besides the mouse. The key to the ES cell technology is the ability to enrich and screen a large number of genomes by drug selection for extremely rare events of gene targeting. To develop this powerful technology in medaka, I have established conditions for efficient gene transfer and drug selection in MES1 cells and demonstrated that medaka ES cells after long-term drug selection retain the developmental potential in vitro and in vivo (Hong et al., 2004a). I have also shown that MES1 cells display totipotency-specific gene expression by demonstrating the ability to activate the mouse Oct4 promoter (Hong et al., 2004b). This offers a promise to develop the ES cell technology in the medaka as a first non-mammalian model. Using the medaka p53 genomic sequence, isogenic HR vectors allowing for PNS were constructed based on the neo and tk cassettes. PNS in MES1 cells resulted in a 10-fold enrichment.

Spermatogenesis is an excellent model for stem cell self-renewal and differentiation. The male germ stem cells spermatogonia are unique adult stem cells because they can produce sperm to transmit genetic information between generations and thus have potential for reproductive engineering. Either establishment of spermatogonial cell lines or in vitro recapitulation of sperm production from such cell lines has long been the target of scientific research. However, normal mammalian spermatogonia without genetic immortalization/transformation cannot be cultured for long. It has been unclear whether spermatogonial cell lines have the intrinsic inability for culture or the culture conditions used were not suitable. Recently, my group has reported two breakthroughs: succeeded a first cell line of normal medaka spermatogonia capable of test-tube sperm production (Hong et al., 2004c). This offers a first opportunity to study male germ stem cells and to recapitulate spermatogenesis in vitro for germline transmission and molecular analyses of human male infertility. These breakthroughs did shock the world scientific community and public as evidenced by a large number of reports/citations by newspapers and other media (e.g., search for 'Singapore test-tube sperm' at 'google' or 'yahoo').

Awards:

Scholarship of 'DAAD' ('German Academician Exchange Program')
Top-100 Talent Scientists of Chinese Academy of Sciences 2000
Outstanding Researcher Award of National University of Singapore (2005)

Patents: Spermatogonial cell line, US patent, 60/553,120, 16 March 2004

Selected publications: