Gene Transfection Into Murine Embryonic Stem Cells And The Regulation Role Of Rac1 In Vessel Forming Of Early Embryonic Development

Background and objectiveNowadays, the most attractive theory and method of therapy in CHD is therapeutic angiogenesis, which is based on vessel forming of early embryonic development to stimulate the growth of new vessels in myocardial ischemia zone and collateral circulation, representing the new direction of therapy in CHD. Embryonic stem cells (ESCs) are pluripotent cell lines of earlier embryonic origin cultivated in vitro with anti-differentiation factors. When cultured in vitro, ESCs can keep undifferentiation state and passage to the future generation. ESCs have similar differentiation potency with earlier embryonic and normal euploid karyotype as their two significant characteristics, so they could be an ideal model for mammal individual development, embryonic differentiation and genetic mechanism of characteristics. Gene transfection engineering is to transfer or transport the nucleic acid having biological function into cells and keep the function in cells, furthermore to utilize in function study of genome, such as gene expression regulation, gene function regulation, signal transduction and drug screening research. In gene transfection aspect ESCs are the received subjects having widely using in gene transfection, localization and integration.However, the practical manipulation has some troubles such as no standards in gene transfection method, huge transfection efficiency variance and difficulty of positive clones screening. When cultured without feeder cells such as mouse embryonic fibroblast(MEF) or in the absence of anti-differentiation factors as leukemia inhibitory factor(LIF), ES cells can spontaneously develop into 3-dimensional, multicellular aggregates called embryoid bodies(EBs), which contains structures of endoderm, mesoderm and ectoderm. As an ideal model for in vitro ESCs differentiation, EBs recapitulate many aspects of the lineage specific differentiation programs and temporal and spatial gene expression patterns of early embryogenesis. Therefore, the EBs system is widely used in investigating early embryonic developmental events such as mutual induction of germinal layers, organ cavitation and so on. The GTPases of RhoA, Rac1 and Cdc42 are the most studied members of the small GTPases, which are molecular switch transducing intracellular signals from growth factors, but it in diffenrentiation of angioblastic cell during early embryogenesis has not been investigated. We observed the growing states of ESCs on different feeder layers and compared the different methods of exogenous gene transfection into ESCs for reseaching transfection efficiency. We established transgenic ESCs lines expressing pcDNA3.1+Rac1G12V and pcDNA3.1+Rac1T17N under the transcriptional control of the CMV promoter, to explore the conditions and characteristics of endothelial cells(ECs) differentiation as well as new blood vessel formation from in vitro differentiation of mouse ESCs, laying a foundation for further studies on mechanisms of blood vessel development.Materials and methods1. Cell Culture Murine R1 ESCs were grown on feeder layers of mitomycin-treated mouse fibroblastic STO or MEF cells in ESCs medium. ESCs were subcultured at semi-confluence, and the medium was changed every day to maintain the cells in an undifferentiated state.2. Plasmid construction The present of pcDNA3.1+Rac1T17N and pcDNA3.1+ Rac1G12V were amplificated, sequenced and linearized for retrieve and purification. The cDNAs encoding Rac1T17N and Rac1G12V get by KpnⅠ/ApaⅠcutting in pcDNA3.1+Rac1T17N and pcDNA3.1+Rac1G12V were subcloned in pPGK-GFP-C1 vector in which the CMV promotor sequence was exised and replaced by the PGK promoter.3. Electroperation of embryonic stem cells After linearization the plasmid was electroporated into 2.5×107 ESCs of the line R1. The ESCs clones were propagated in the presence of LIF 1000 u/ml and selected for 10 days using G418. Pick the 1.5~2 mm drug-resistant ESCs clones that formed after about 10 days of selection with a drawn-out Pasteur pipette under a dissecting microscope.4. Genomic PCR for screening ESCs stable clones PCR amplification on genomic DNA was carried out with the Taq PCR master mix. For PCR detection of the transgene, we used a pair of primers that anneal to the region of the neomycin cDNA in pcDNA3.1+ vector.5. ESCs differentiation EBs were generated from modified ESCs using protocols as described previously. Briefly, subconfluent ESCs were dispersed with 0.25% trypsin-0.53 mmol/L EDTA and plated onto gelatin-coated dishes for 3h to allow feeder cells to selectively attach. Nonadherent ESCs aggregates were then dispersed and cultured on bacteriological petri dishes in ESCs medium without LIF. ESCs were cultivated in hanging drops for 6 days, afterwards plated onto 0.1% gelatin -coated surfaces and allowed to attach in DMEM/10% FCS. Medium was changed on a daily basis.6. EBs Immunohistochemistry Fixed EBs were washed with 0.05 M TBS and permeabilized with 0.25% Triton X-100 and 0.5 mmol/L NH4Cl in 0.05 mmol/L TBS. Before incubation with antibodies, EBs were blocked with 5% bovine serum albumin in TBS. Primary antibodies were anti-mouse SMα-actin and CD31 used at 1:200 dilution. Negative controls were performed without primary antibodies. Detection was performed with a red-fluorescence-labeled secondary antibody.7. RNA extraction and RT-PCR. RNA was extracted from EBs at a variety of time points with Trizol. Reverse transcription was carried out on 1μg of total RNA : Rac1, CD31, SMα-actin, GAPDH.8. SDS-PAGE electrophoresis and Western blot analysis. Western blot was used to observe the changes of Rac1 both in ESCs and EB after transfection using pull down analysis. Proteins were assayed using the Bradford reagent, and the same amount per well was run in 12% SDS-PAGE and transferred to a nitrocellulose membrane.Blots were probed with a polyclonal anti-Rac or others, and a secondary peroxidase-conjugated antibody. Immunoreactive proteins were revealed by ECL.Results1. Morphological characteristics of MEF and ES cells. MEF cells were long spindle like cells and grow to trabs or swirl shape; STO cells were short spindle like and have well-arranged shape and distinct edge. ESCs could maintain undifferentiated on either STO feeder cells or MEF feeder cells, which formed compact cell colony with smooth margin and undistinguished cell boundary. But there were also some differences. When growing on STO feeder cells, ESCs grow relatively slow and formed round colonies mostly; While on MEF feeder cells, ESCs grow rapidly and formed oval colonies.2. Formation of EBs. Numerous EBs were formed 3 days after attached culture of ES cells, and EBs formation efficiency was about 40% on STO feeder and only near 10% on MEF feeder. Endodermal differentiation was easily seen in some EBs of STO feeder but rarely seen of MEF feeder .3. Vector construction and positive ESCs clone screening. New vector pPGK-EGFP-Rac1G12V and pPGK-EGFP-Rac1T17N were identified by KpnⅠ/ApaⅠcutting and sequencing. After electroporation G418-resistant colonies were visible macroscopically after 10 days of culture. We amplified 22 clones of ESCs and 3 of them in each group were confirmed to be positive by genomic PCR. The EGFP positive clonies were visible after 24h transfection by Lipofictamine 2000. But there was higher transfection efficiency of ESCs on MEF feeder about 60.7±11.4% comparing with it on STO feeder of 10.8±7.5%(p<0.05).4. RT-PCR and Western blot to analysis Rac1 expressing in ESCs and EBs. It showed that mRNA level had no difference among Rac1T17N,Rac1G12V and R1 ESCs.In Rac1G12V EBs, Rac activity was increased by near 3~4 fold, whereas Rac activity was abolished in Rac1T17N cell line.5. EBs model establishment and morphologic characteristics. We cultured transfected ESCs to establish EBs model simulating vasculogenesis and angiogenesis processes and founded that Rac1G12V ESCs can develop to typical EBs with three germinal layers like wild-type ESCs R1 in 7~9 days. But Rac1T17N ESCs can not develop in same time. The cells in cystic cavity of Rac1G12V EBs had earlier apoptosis and appearing of CD31 positive cells whereas had not this finding of Rac1T17N EBs.6. Observation of EBs in attached differentiation. Two transfetion EBs were plated onto cover slips coated by 0.1% gelatin for attached differentiation. In the differentiation system EBs of Rac1G12V showed active differentiation and migration. It showed complete cystoskeleton, well-distributed stress fiber and migratory cells out of EBs which stained CD31 and actin positive.However, EBs of Rac1T17N had hindered differentiation and migration. It showed less stress fiber concentrating near cell membrane and no migratory cells appearing. RT-PCR and Western blot showed early expressing of CD31 RNA and protein in Rac1G12V EBs whereas less and later in Rac1T17N EBs. DiI-Ac-LDL labeling staining of differentiated cells were observed in Rac1G12V for blood vessel like structure formation but Rac1T17N EBs were not.ConclusionClones of Rac1G12V and Rac1T17N expressing in embryonic stem cells were successfully established for simulating angiogenisis process in early embryonic development. Rac1 was the necessary moderating gene in that process, controling cell apoptosis of cystic cavity, ECs genesis and migration. Lipofectamine 2000 had higher transfection efficiency in ESCs. MEF feeder was the first choise for Liposome transfection and STO feeder was feasible for G418 screening and EBs plating.