2. Material and methods
2.1. cDNA cloning and plasmids construction
The cDNAs of reprogramming inducing factors Oct4, Sox2, Lin28 and Nanog were amplified by PCR, using Pfu DNA polymerase (Clontech, USA). cDNA from hES cells was used as the template to amplify Lin28 and Nanog. A plasmid containing Oct4 (Clone ID: 40125986, Open Biosystems, USA) and a plasmid containing Sox2 (Clone ID: 2823424, Open Biosystems, USA) were used as the template to amplify Oct4 and Sox2, respectively. A 9-amino BI6727
(RKKRRQRRR) membrane-penetrating domain (MPD) from the TAT protein of HIV was
added at the N-terminus using modified sense primers, shown in Table1.
Primers used for cloning.PrimersSequencesGenBank Acc. No.Lin28-F5-GGAATTCCCGCAAGAAGCGCAGACAGCGCCGTCGAGGAGGCGGTGGGATGGGCTCCGTGTCCAACCAG-3NM_024674Lin28-R5-ATAGCGGCCGCTCAATTCTGTGCCTCCGGGAG-3NM_024674Nanog-F5-TCCCCCGGGGGAACGCAAGAAGCGCAGACAGCGCCGTCGAGGAGGCGGTGGGATGAGTGTGGATCCAGCTTGTC-3NM_024865Nanog-R5-ATAGCGGCCGCTCACACGTCTTCAGGTTGCATG-3NM_024865Oct4-F5-TCCCCCGGGGGAACGCAAGAAGCGCAGACAGCGCCGTCGAGGAGGCGGTGGGATGGCGGGACACCTGGCTTCG-3BC117435Oct4-R5-ATAGCGGCCGCTCAGTTTGAATGCATGGGAGAGC-3BC117435Sox2-F5-TCGCCCGGGCGAACGCAAGAAGCGCAGACAGCGCCGTCGAGGAGGCGGTGGGATGTACAACATGATGGGAGCG-3BC013923Sox2-R5-ATAGCGGCCGCTCACATGTGTGAGAGGGGCAG-3BC013923Note: The underlines indicated the restriction enzyme cutting sites.Full-size tableTable optionsView in workspaceDownload as CSV
The PCR products were then sub-cloned into pCR4-TOPO vectors (Invitrogen, USA). After sequence verification, they were restricted with EcoRI/SmaI/SrfI and NotI, followed by sub-cloning into the equivalent sites of the baculovirus transfer plasmid, pBAC-3 (Novagen, Nottingham, UK). The resulting clones were then verified by sequencing, and were referred to herein as pBAC-3/TAT-gene.
Sf9 insect cells were adapted to growth in suspension or monolayer in Sf-900 II SFM (Life Technologies, Basel, Switzerland). Suspension culture cells
were usually seeded at 0.5uu106ucells/mL in a total volume of 50umL in a 250-mL disposable plastic Erlenmeyer flask. Exponentially growing cells were incubated in a temperature-controlled orbital shaker at 28uC, 150urpm. Cells were split when the density reached 4uu106ucells/mL. Typically, cells grown at 28uC in a monolayer were split 1:8 every 3a4udays, if the ###http://www.amino-11-ddutp.com/image/1-s2.0-S2093791110120046-gr1.jpg####cells
were healthy and confluent (85a95%). Cell density and viability were determined by the Trypan Blue exclusion method.
2.3. Expression of fusion proteins
A BacMagic Transfection Kit (Novagen, Nottingham, UK), including BacMagic DNA, Insect GeneJuice Transfection Reagent, a Transfection Control Plasmid, Sf9 Insect Cells, and BacVector Insect Cell Medium, was used for expression of fusion proteins, according to the recommended protocol. Instead of a recombinant transfer plasmid, a corresponding amount of medium and 500ung of the supplied Transfection Control Plasmid were used as negative and positive transfection controls, respectively. After 5udays of incubation, medium containing seed stock of the recombinant baculovirus was harvested. The virus was amplified in a suspension culture at a low multiplicity of infection (MOI), according to the recommended protocol. When cells appeared to be well infected with virus (usually 3a5udays), the cell culture medium was harvested by centrifugation at 1000ug for 20umin at 4uC. The supernatant was removed aseptically and stored (recombinant virus) in dark at 4uC, or at u80uC for long-term preservation. The virus titer was quickly determined by the help of FastPlax™ Titer Kit (Novagen, Nottingham, UK) before the virus was used in subsequent experiments.