Cells will adhere as usual in the presence of complexes.
Plat e cells transfection protocol.
The first recombinant plant derived pharmaceutical protein human serum albumin was initially produced in transgenic tobacco and potato plants the delivery and expression of recombinant dna in living cells have proven invaluable in a wide variety of applications for basic plant research plant biotechnology and molecular farming.
18 24 hours prior to transfection plate phoenix cells at 1 5 2 million cells per 6 cm plate in producer cell growth media.
Plat e cells contain gag poland envgenes allowing retroviralpackagingwith a single plasmid transfection.
This distributes cells evenly about the plate.
Cloned genes can be transfected into cells for biochemical characterization mutational analyses investigation of the effects of gene expression on cell growth investigation of gene regulatory elements and to produce a specific protein.
The platinum retroviralpackaging cell lines are based on the 293tcell line.
These cells require transfection of only an expression vector to produce retrovirus.
High retroviral yields with plat e cells.
Transfect a platinum retrovirus packaging cell line such as plat a amphotropic or plat e ecotropic which stably expresses both mmlv gag pol and an amphotropic or ecotropic envelope protein with a retroviral expression vector.
After quickly thawing the cells in a 37ºc water bath immediately transfer the thawed cell suspension into a 15 ml tube containing 10 ml of culture medium.
Surface areas may vary depending on the manufacturer.
Establishing plat a cultures from frozen cells 1.
Prepare complexes in the plate and directly add cells at twice the cell density as in the basic protocol in a 100 µl volume.
You may perform rapid 96 well plate transfections by plating cells directly into the transfection mix.
After quickly thawing the cells in a 37ºc water bath immediately transfer the thawed cell suspension into a 15 ml tube containing 10 ml of culture medium.
After adding cells set plates in incubator and gently shake forward and backward then side to side 3 4 times.
They exhibit longer stability and produce higher yields of retroviralstructure proteins.
The platinum e plat e cell line a potent retrovirus packaging cell line based on the 293t cell line was generated using novel packaging constructs with an ef1α promoter to ensure longer stability and high yield retroviral structure protein expression gag pol ecotropic env.
