Cell Lines

Cell Lines

1. Mammalian Cells

   • Capacity of proper protein folding, assembly and post-translational modification.
     • Better host for recombinant proteins
       • Pharmaceuticals potential
         • Production for recombinant cells
           • Cultivation process is determined by production needs.
           • Recombinant gene with transcriptional regulatory elements present in cells.
             • Selective agent is added to gene.
               • Selection occurs in the absence of several metabolites
                 • Prevents growth of nontransformed cells
               • DHFR, GS, among others
               • Expression of selective genes in survivor cells.
                 • Transferred as single cells to cultivation vessel,.
                   • Cultures are expanded to produce clonal populations.
                     • Individual clones with high potential are used for cultivation and analysis.
                       • One cell line with suitable growth and productivity is selected for production.
               • Selective gene can be derived from a weak promoter.
                 • Possibility of obtaining high-level producer cell lines.
                   • It might reduce the efficiency of stable transfection
                     • Survivor cells yield to more recombinant product.
                     • Linearization of plasmids before transfection.
                       • Improve a stable transfection
                         • After 1-2 hours
                           • Supercoiled plasmid DNA
                             • Converted into linear molecules with nucleus.
                               • Due to activity of endo and exonucleases.
           • Non viral gene transfer is used to generate stable cell lines
             • Calcium phosphate transfection, electroporation, lipofection and biolistic and polymer-mediated gene transfer
               • Efficient & reliable.
         • Understanding of gene expression, metabolism, growth and apoptosis delay.
           • Improvements in vectors, host cell engineering, medium development and screening methods.
       • DNA delivery and integration
         • Expression vectors
           • Used for recombinant cell line generation
             • Gene of interest is isolated as a cDNA without introns.
             • Viral or cellular promoter/enhancer to drive expression of recombinant gene.
               • Eukaryotic cells
                 • Cytoplasmic transport and translation of mRNA depends on splicing.
               • strong viral promoters from cytomegalovirus (CMV) and SV40
                 • Many commercial vectors use EF-1alfa promoter although expression from viral promoters
           • Include one intron sequence at least
             • Located between promoter and cDNA coding sequence.
               • Modifying the coding region may improve the level of expression
           • Recombinant gene and selection gene
             • Can be expressed from a poly cistronic mRNA
             • Gene silencing
               • Correlates with different factors
                 • Histone hypoacetylation
                 • Methylation of lysine 9 of Histone H3
                 • Increase in CpG methylation in the promoter region of transgene
       • Site of integration of recombinant gene
         • Position effect
           • Major effect on the transcription rate.
             • Heterochromatin
               • Integration in inactive regions results in little or no transgene expression.
             • Euchromatin
               • Ingration into active regions allows transgene expression
                 • might not be sufficient to ensure long-term expression of recombinant gene.
   • CHO, NSO, BHK, HEK-293 and human retinal cells.

2. Insect Cell

   • Baculovirus
     • Principal method for production of challenging cytosolic prokaryotic that are unable to be effectively synthesizes in prokaryotic hosts.
       • Costly as mammalian cell culture
         • cultured in serum containing classical media, chemically defined media or serum-dree media
           • streamlined in development of recombinant viral backbone using bacterial homologous recombination, viral production and amplification
             • still represent time-consuming steps prior to protein expression trails
               • Construction, amplification, tittering and optimization are lengthy steps and often require a month or more
     • unable to produce sialylated complex glycans
       • dependence on viral transduction presents fundamental issues that amplified considering over-expression of secreted proteins

3. Yeast cells

   • Advantages
     • less expensive than insect and mammalian cells
       • high density in reusable glass Erlenmeyer
         • time relative low
           • cell doubling is rapid
             • this make the expression experiments relatively straight-forward and quick
   • Disadvantages
     • Express a number of proteases
       • lead to significant yet often protein dependent and protein degradation

4. Transient Expression

   • Biochemical characterization
   • Consumable reagents
   • Fast analysis of protein variants

5. Episomal Expression

   • base on viral elements promote autonomous replication and retention in the nucleus