Відмінності між версіями «As described previously, Alca binds to KLC to activate kinesin-1's association with Alcacontaining vesicles, and Alca's WD motif is enough to recruit kinesin-1 to these vesicles to activate their anterograde transport»
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| − | + | Cells have been infected with either the SSEA4 or CD24 antibody conjugated m 168 pseudotyped virus bearing the pSin-EF2-TKPuro lentiviral vector, five days post BMP4 addition and counterselected with two mM ganciclovir. Trophoblast formation was monitored for expression of cytokeratin 7, a marker of both villous and extravillous trophoblasts by flow cytometry ten days post BMP4 addition. The percent cells expressing cytokeratin 7 elevated because of ablation of the cells expressing the TK gene by way of either the CD24 or SSEA4 antibody mediated m 168 lentiviral gene delivery. Within the absence of treatment with Ab-targeting virus, 9.9% on the cells differen- Targeted Gene Delivery to Human ES and iPS Cells tion, this isn't a concern. For every technique, the shortfalls are countered by their benefits. The usage of the antibody-conjugated m 168 pseudotyped virus has the prospective to both strengthen the selection and identification of iPS cells, leading to insights into their reprogramming, also as the research of hES cells, where delivery of regulatory proteins and selectable markers can increase the homogeneity in the pathways of interest. Supplies and Approaches Cell culture Human H9 ES cell line was in the WiCell Research Institute. iPS5 cells had been a gift from Dr. George Q. Daley. hES and iPS5 cell lines had been maintained in feederfree cultures on Matrigel-coated six-well plates with mTeSRTM1. For regular passage, hES cells have been treated with 1 mg/mL of dispase for five min, collected having a cell scraper and plated. For virus transduction, cells were treated with 1 mg/mL Accutase for five ten min till the colonies had been dissociated into single cells. The single-cell colonies were then detached and plated with mTeSRTM1 containing 5 mM ROCK-inhibitor Y27632 for 24 hr. HEK293T cells purchased from American Kind Culture Collection and maintained in Dulbecco's modified Eagle medium containing 10% fetal bovine serum and antibiotics & antimycotics. Plasmids construction Two vectors had been utilized that encoded either the cytomegalovirus promoter or the human elongation factor-1a promoter to drive the GFP expression. The CMV-GFP expression vector, pHR'CMVGFPW, was kindly provided by Dr. J. Dougherty. pSin-EF2-Oct4-Puro, encoding the EF1apromoter was modified to express either GFP or herpes simplex virus thymidine kinase gene cassettes by way of replacement of the Oct4 gene from SpeI to EcoR1. The GFP gene was amplified using pGIP as template with the following primers: EF1aGFP fwd, 59-GCA CTA GTG CCA CCA TGG TGA GCA AGG GCG AG -39; EF1a-GFP rev, 59-GGC GAA TTC TTA CTT GTA CAG CTC GTC CAT GCC -39. The TK gene was amplified from the template pAL120-TK using the [http://www.gliderjockey.com/members/design0poppy/activity/236563/ Two central pathways for generation of ceramide in apoptosis are de novo synthesis beginning with condensation of palmitoyl-CoA to serine, catalyzed by serine palmitoyltransferase, and hydrolysis of sphingomyelin by sphingomyelinases] primers EF1a-TK fwd, 59- AGC ACT AGT GCC ACC ATG GCT TCG TAC CCCTGC-39 and EF1a-TK rev, 59-GGC GAATTC TCA GTT AGC CTC CCC CAT CTC -39. The SpeI and EcoRI restriction sites are underlined. All with the constructs were verified by DNA sequencing. The chimeric Sindbis viral envelope vector m 168 was a gift from Dr. I. Chen . The HIV-1 packaging vector pCMV-dR8.2dvpr was purchased from Addgene. The plasmid pHIT-G expresses VSV-G. Human iPS cells generation Human iPS cells had been induced by retroviral particles which had been produced by co-transfection from the retroviral pMXs vector individually expressing the 5 transcription factors, including Oct4, Sox2, c-Myc, Nanog, and Klf4 plus | |
Поточна версія на 08:32, 10 травня 2017
Cells have been infected with either the SSEA4 or CD24 antibody conjugated m 168 pseudotyped virus bearing the pSin-EF2-TKPuro lentiviral vector, five days post BMP4 addition and counterselected with two mM ganciclovir. Trophoblast formation was monitored for expression of cytokeratin 7, a marker of both villous and extravillous trophoblasts by flow cytometry ten days post BMP4 addition. The percent cells expressing cytokeratin 7 elevated because of ablation of the cells expressing the TK gene by way of either the CD24 or SSEA4 antibody mediated m 168 lentiviral gene delivery. Within the absence of treatment with Ab-targeting virus, 9.9% on the cells differen- Targeted Gene Delivery to Human ES and iPS Cells tion, this isn't a concern. For every technique, the shortfalls are countered by their benefits. The usage of the antibody-conjugated m 168 pseudotyped virus has the prospective to both strengthen the selection and identification of iPS cells, leading to insights into their reprogramming, also as the research of hES cells, where delivery of regulatory proteins and selectable markers can increase the homogeneity in the pathways of interest. Supplies and Approaches Cell culture Human H9 ES cell line was in the WiCell Research Institute. iPS5 cells had been a gift from Dr. George Q. Daley. hES and iPS5 cell lines had been maintained in feederfree cultures on Matrigel-coated six-well plates with mTeSRTM1. For regular passage, hES cells have been treated with 1 mg/mL of dispase for five min, collected having a cell scraper and plated. For virus transduction, cells were treated with 1 mg/mL Accutase for five ten min till the colonies had been dissociated into single cells. The single-cell colonies were then detached and plated with mTeSRTM1 containing 5 mM ROCK-inhibitor Y27632 for 24 hr. HEK293T cells purchased from American Kind Culture Collection and maintained in Dulbecco's modified Eagle medium containing 10% fetal bovine serum and antibiotics & antimycotics. Plasmids construction Two vectors had been utilized that encoded either the cytomegalovirus promoter or the human elongation factor-1a promoter to drive the GFP expression. The CMV-GFP expression vector, pHR'CMVGFPW, was kindly provided by Dr. J. Dougherty. pSin-EF2-Oct4-Puro, encoding the EF1apromoter was modified to express either GFP or herpes simplex virus thymidine kinase gene cassettes by way of replacement of the Oct4 gene from SpeI to EcoR1. The GFP gene was amplified using pGIP as template with the following primers: EF1aGFP fwd, 59-GCA CTA GTG CCA CCA TGG TGA GCA AGG GCG AG -39; EF1a-GFP rev, 59-GGC GAA TTC TTA CTT GTA CAG CTC GTC CAT GCC -39. The TK gene was amplified from the template pAL120-TK using the Two central pathways for generation of ceramide in apoptosis are de novo synthesis beginning with condensation of palmitoyl-CoA to serine, catalyzed by serine palmitoyltransferase, and hydrolysis of sphingomyelin by sphingomyelinases primers EF1a-TK fwd, 59- AGC ACT AGT GCC ACC ATG GCT TCG TAC CCCTGC-39 and EF1a-TK rev, 59-GGC GAATTC TCA GTT AGC CTC CCC CAT CTC -39. The SpeI and EcoRI restriction sites are underlined. All with the constructs were verified by DNA sequencing. The chimeric Sindbis viral envelope vector m 168 was a gift from Dr. I. Chen . The HIV-1 packaging vector pCMV-dR8.2dvpr was purchased from Addgene. The plasmid pHIT-G expresses VSV-G. Human iPS cells generation Human iPS cells had been induced by retroviral particles which had been produced by co-transfection from the retroviral pMXs vector individually expressing the 5 transcription factors, including Oct4, Sox2, c-Myc, Nanog, and Klf4 plus
