Stable suppression of gene expression in murine embryonic stem cells by RNAi directed from DNA vector-based short hairpin RNA.

Murine embryonic stem (ES) cells are an ideal system for the research of directed differentiation in vitro. Long double-stranded RNA, which can induce RNA interference (RNAi) effectively in many organisms, has been shown to suppress target gene expression efficiently and specifically in undifferentiated ES cells. However, it cannot be used in differentiated ES cells due to unspecific inhibition of gene expression resulting from the activation of interferon pathway following differentiation. Using green fluorescent protein (GFP) as a reporter system, we show here that a short hairpin RNA (shRNA) expression vector driven by the murine U6 small nuclear RNA promoter can specifically induce potent gene knockdown effect (i.e., inhibit GFP expression specifically) when transfected transiently into ES cells. Furthermore, when the expression vector is stably integrated into the genome of the cell, it can still show specific RNAi effect, which can be maintained at least for 10 days. These transfected ES cells showed no obvious differences in the morphology or growth rate in culture compared with untransfected cells, suggesting that the activation of shRNA-directed RNAi did not affect the properties of ES cells and that the RNAi effect in ES cells is specific and persistent. Our results prove the feasibility of the U6 promoter-driven shRNA expression technique to be used to study the function of genes expressed in ES cells. These ES cells, after integration of the U6-based RNAi vector into their genome, could be used to generate gene knockdown mice.

Comparison of the method establishing embryonic stem cell lines from five different mouse strains.

We compared the characteristics of the method establishing embryonic stem cell lines from five different mouse strains using the medium containing 70% rat heart cell-conditioned medium (RH-CM) as ES cell culture medium, using the primary murine embryo fibroblast as feeder cells, and using the digestive enzyme buffer containing 1% chicken serum and "the series digestive method". We first reported new ES cell lines established from the outbred strain mice KM and ICR using the improved method in our lab and the ratio of establishment of ES cell lines from KM and ICR strain mice is up to 12% and 42.1% respectively. Compared with routine method of establishing ES cell lines, the improved method made distinct differences, increasing the ratio of ES cell line's establishment of 129/ter mouse from 11.8% to 33.3%, that of C57BL/6J mouse from 3.7% to 13.3%, that of BALB/c mouse from 2.9% to 19.4%. We tested the appropriate dispersing occasion, that is proliferating period of the ICM, affected the formation of ES clones and the ratios of ES cell lines established. It was shown that the most appropriate dispersed occasion for the ICM of 129/ter, C57BL/6J, BALB/c, KM and ICR mice was 4-6 d, 3-3.5 d, 4 d, 4-5 d, 4-5 d after ICM proliferation respectively. At the same time, the effects of the concentration of digestive enzyme buffer were discussed. It was found that the ES cells from BALB/c mice were sensitive to the high concentration of digestive enzyme buffer and the 0.05% Trypsin-0.008% EDTA is an ideal concentration for their establishment and maintenance. It was shown that 'the series dispersed method' was much better than 'the once dispersed method' on the aspect of dispersing the proliferating ICM and formation of ES clones. Compared with the routine ES cell culture medium containing mLIF, the RH-CM not only remarkably inhibited the differentiation of murine ES cells and maintained their diploid karyotype, but also promoted the attachment and growth of ES cells. This improved method of establishment and culture of ES cell lines effectively maintained a series of their characteristics of pluripotent embryonic stem cells.

[RNA interference directed by small hairpin RNA expressed in COS-7 cells].

RNA interference is a phenomenon of gene silencing directed by double-stranded RNA. It can specifically inhibit gene expression by degrading mRNA efficiently and has been widely used to knockdown gene expression in Caenorhabditis elegans, Drosophila melanogaster, etc. For mammalian cells, dsRNA directed RNAi was detected only in murine undifferentiated ES or embryonic carcinoma (EC) cells. Our previous work proved the existence of RNAi effect for reporter gene GFP and endogenous gene Oct4 in undifferentiated murine ES cells. Yet in other kinds of mammalian cells, because of the existence of interferon pathway, long dsRNA will induce the cells to shutdown global protein translation and go to apoptosis. Therefore, dsRNA longer than 30 bp cannot be used to induce specific gene knockdown effect in these cells. Elbashir et al found that in vitro synthesized small interfering RNA (siRNA) (19-23 nt) could induce potent RNAi as effective as long dsRNA without showing unspecific effect, so that the interferon pathway could be bypassed. It was shown that during RNAi process, long dsRNA was first degraded into 19-23 nt siRNA and then recruited into RISC (RNA induced silencing complex) to degrade corresponding mRNA. However, the synthesis of siRNA is expensive and the effect is transient because the knockdown effect can only be maintained for about a week. Recently, it has been shown that U6 promoter directed small hairpin RNA (shRNA) can induce potent gene knockdown effect in murine P19 Embryonic Carcinoma cell. The RNAi effect of U6 promoter-driven shRNA corresponding to Green Fluorescence Protein (GFP) in COS-7 cells was checked. And it was found that the U6 promoter-driven shRNA for GFP can specifically and potently knockdown the GFP's expression in COS-7 cells. The result established the feasibility of using RNAi technique directed by U6 promoter-driven shRNA to study genes' function in COS-7 cell line.

[RNA interference in three ES cell lines from different mouse strains].

RNA interference phenomenon in three different murine ES cell lines (MESPU13, B3, and R1) is reported. A vector(pdsGFP) was used that transcribed hairpin double-stranded RNA of GFP gene to transfect ES cells by using lipofectin. The transient transcription of dsRNA induced RNAi (RNA interference) in the ES cells. That is, the double-stranded RNA of GFP gene potently turned down the expression of the GFP gene. On the hand, the linearized plasmid pdsGFP-puro was electroporated into MESPU13 ES cells, and the expression level of GFP after puromycin screening was turned down obviously in about 30% ES cell clones; and in a few clones, the expression level of GFP was not observed under the fluorescence microscope and GFP mRNA was not detectable by RT-PCR. Further more, another vector (pdsOCT4) was constructed that transcribed double-stranded RNA of OCT-4 gene which is specifically expressed in ES cells. ES cell clones that stably integrated the vector were screened after the electrotransfection of the cells with the above construct. 51 random-selected clones were amplified and 48 of them were checked by semi-quantitative RT-PCR. In 11 of them the mRNA of OCT-4 was undetectable by RT-PCR. This means that RNAi can be used to study mammal and human gene's function in ES cell lines from different strain mice.

[Current state of establishing and maintaining human embryonic stem cell lines and key problems in these studies].

Derivation of human embryonic stem cell and embryonic germ cell lines has widespread and far-reaching significance on human basic research and transplantation therapies. Human pluripotential stem cells provide an exciting new model for studying early human embryogenesis, understanding normal human development and abnormal development, provide a powerful system for discovering human novel genes and testing their function, offer new strategies for discovering of novel growth factors and medicines and promise a renewable source of cells for tissue transplantation, cell replacement and gene therapies. Research history of establishment of human ES and EG cell lines is reviewed. Several methods of establishment of these cell lines involving in the protocol, route, significance and possibility are discussed. Selection of the feeder layer, medium, and supplemental cytokines and their roles in establishing and maintaining human ES and EG cell lines at present are illustrated in detail systematically. Effects and used methods of several kinds of digestive en-zyme in propagations are prepared. Several methods for identifying human ES and EG cells are summarized. At the end, some key problems which are urgent to resolve in these studies at present are put forward and analyzed.

[Researches on the methodology of establishment of BALB/c murine embryonic stem cell lines].

Several methods and processes of establishment and culture of BALB/c mouse ES cell lines were discussed detailedly. A new method to establish and culture ES cell lines derived from BALB/c mouse was set up successfully using mouse embryonic fibroblast feeder layer and rat-heart-cell-conditioned medium (RH-CM). These culture conditions not only maintain the undifferentiated state and normal diploid karyotypes of BALB/c mouse ES cells effectively, but also maintain a series of their characteristics of murine stem cells. two different kinds of digestive methods and Two kinds of digestive juice with different concentrations were designed to dissociate proliferous inner cell mass (ICM) and ES cell colonies derived from dissociative ICM. two different kinds of digestive methods are "single time dissociation method" and "several times dissociation method", two kinds of digestive juice are 0.25% Trypsin-0.04% EDTA and 0.05% Trypsin-0.008% EDTA. At the same time, appropriate dissociated occasion of ICM and the effect of RH-CM on establishment and culture of BALB/c mouse ES cell lines were discussed. The results suggested that it is a reasonable method to establish BALB/c mouse ES cell lines using low concentration digestive juice and "several times dissociation method" to dissociate ICM after 4 days' proliferation. Judged by the form of ES cells and its colonies, proliferous capability, karyotypes examine, alkaline phosphatase activity assay and differentiation capability in vitro and in vivo, the 9 ES cell lines that we established satisfied the all traits of murine ES cell line.

[Germ cells in the murine embryonic development].

This paper reviewed the recent progress of the origin, migration and proliferation, sex determination, and genomic modification of murine germ cells during its embryonic development. Murine germ cells originate from primordial germ cells at about 7-7.5dpc. Then PGCs migrated into germinal ridge at about 12.5dpc during which Steel/c-kit signal pathway plays important roles and stopped division at 13.5dpc. The sex of germ cells was mainly determined by the soma microenvironment in the gonad. And there are essential genes for sperm formation on the Y chromosome. The de novo methylation of murine germ cells was much later than soma cells and was completed at about 18.5dpc. The X chromosome reactivation of female germ cells was finished at about 14.5-15.5dpc which was independent of sexual differentiation of germinal ridge.

[Discussion of the methods for establishing embryonic stem cell lines from 129/ter. C57BL/6J mice with high efficiency].

A new method for establishing ES cell lines from 129/ter. C57BL/6J mice was set up which was characterized by the murine embryonic fibroblast cell(MEF) feeder, the medium of rat heart cell-conditioned medium(RH-CM) for ES cells, and the consecutive digestion by the digestion liquid containing 1% serum. Every group of improved experiments was done with a control of routine method. The results showed that, compared with routine method, the improved way increased the ratio of ES cell lines of 129/ter mice from 11.8% to 33.3%, and of C57BL/6J from 3.7% to 13.3%. The difference is distinct. The passage culture of ES cells showed that, compared with medium added LIF, RH-CM not only inhibited the differentiation of murine ES cells, maintained its dipoild karyotype, but also promote its adherence growth. This kind of culture condition not only maintained the ES cells in an undifferentiated state and their normal dipoild karyotype, but also a series of other characteristics of totipotent embryonic stem cells during extended culture period.