[Formation of germline chimeras from murine embryonic stem cell lines].

Generation of germline chimeras is the crucial step in ES cell-mediated transgenesis. The prerequisite for germline chimerism is the maintenance of germline differentiating potency of ES cells, whereas production of germline chimeras is the only method to prove whether such potency is maintained. In order to investigate the germline differentiating potency of three newly established ES cell lines (MESPU21, MESPU22 and MESPU29), ES cells were introduced into host embryos from inbred C57BL/6J and outbred KMW or ICR through blastocyst injection or 8-cell stage morula injection. Totally 81 chimeras were obtained; among 42 test-bred ones, 19 were germline transmitters assessed by coat analysis, as is the first report of ES cell-embryo germline chimeras in China. MESPU21 and MESPU22 formed germline chimeras in high frequency and most of those chimeras produced ES cell-derived progeny in high proportion, which proved that both ES cell lines retained good germline differentiating potency and could be used as valuable cellular vehicles to introduce genetic modifications into mouse genome.

[The specific labelling of mouse embryonic stem cells].

Despite the wide application of ES cell technology, little is known about the pluripotent nature of ES cells. This is partly due to the heterogeneity of ES cell population in culture. This report described the specific labelling of undifferentiated cells in ES cell lines. oct-4 gene is specifically expressed in all the totipotent cells in mouse embryos and undifferentiated ES cells. A constructed pG18NG was obtained by inserting the reporter gene beta geo into the oct-4 transcription elements. ES cell lines MESPU22 and MESPU13 were transfected with this construct and stable integrated cell clones were selected out. With the experiments of in vitro cultivation, differentiation and chimeras production, it was confirmed that we have successfully labelled the undifferentiated cells in ES cell lines, and this label was both valid in vitro and in vivo.

[Establishment of two ES cell lines with good germline contribution].

Nine embryonic stem cell lines have been established from mouse strain 129/ter. Three of the nine ES cell lines were karyotypically normal. The nine cell lines showed some difference in the growth rate and the differential competence. Chimeras were made by injecting the ES cells into C57BL/6J blastocysts, and the germline compositions of the chimeras were detected by mating them with albino ICR mice. The results indicated that ES cell line MESPU21 and MESPU22 were both highly germline-competent. Comparing with other ones, these two cell lines both were karyotypically normal and propagated fast. The tissue composition of the teratocarcinomas derived from these cell lines appeared paralle to the results of chimera production. Careful manipulation during the process of ES cell establishment was helpful to obtain good cell lines.

[Establishment of a high germline competent C57BL/6J ES cell line].

Eleven ES cell lines from C57BL/6J mice were established, with the primary culture of mice embryos as feeder cells and 1 x 10(3) units Leukemia Inhibitory Factor (LIF) in the DMEM (high glucose) medium. The frequency of establishment was 9.6%. Five of these ES cell lines were demonstrated karyotypically normal with diploid composition of over 70%. They shared the characteristics of early embryonic cells: positive for alkaline phosphatase activity and oct4 gene expression. They also showed high potency to differentiate into wide types of cells in vivo. Following injection of the blastocysts, three of them showed abilities to give rise to chimeras and MESPU35 cell line was identified to have high efficiency of colonization into the germline. The clones of MESPU35 cells maintained the germline competence and the mutant clone also retained the high potency to participate into the development of embryos. MESPU35 cells can serve as a valuable vehicle for the production of mutant mice.

[The medium conditioned incubation with rat heart cells maintains the properties of ES cells].

17 kinds of conditioned media were selected by plating tests, propagation and other tests with two cell lines, C-19-2 and MESPU-13. The results suggested that the rat heart cells conditioned media (RH-CM) can inhibit the spontaneous differentiation effectively, maintain the normal diploid karyotypes and promote adherence and proliferation of mouse ES cells. The ES cells were propagated in RH-CM to the 20th passage remaining their pluripotent in vivo and in vitro differentiation ability. RH-CM can be used as supplement of ES cell media. ES cells which are cultured in media containing 70% RH-CM and on PMEF feeder can maintain their undifferentiation state and diploid karyotype. RT-PCR detection suggested that there was mLIF expression in rat heart cells.