Non Viral Transfection Of Mammalian Cells Biology Essay
As ‘process is merchandise ‘ is a cardinal factor in the biopharmaceutical industry, the demand of better techniques for DNA transportation is indispensable for the large-scale recombinant protein production in mammalian cells. Transeunt transfection in mammalian cells such as human embryologic kidney 293 or Chinese hamster ovary cells has been reported for the production of mg to gram measures of recombinant protein.
Calcium phosphate precipitation ( CaPi ) and polyethyleimine ( PEI ) are the two most extensively used non-viral cistron bringing systems. The highest transeunt outputs of 80-100mg/L have been obtained by transfecting CHO cells with PEI of 25 kDa. Non-viral bringing systems have made important advancement in developing better look vectors, pick of cell lines, civilization medium, transfection parametric quantities and procedure scale-up.
Introduction
Stable cistron look engineerings have been widely employed for the production of recombinant proteins from the mammalian cells. Owing to the drawbacks of these techniques ( investing in clip, resources ) and increasing demand of the recombinant proteins in the market, it has become apparent to develop a technique that is faster, cheaper and more dependable. With the coming of the transeunt systems in 1990s, many plants have been done for the production of mg to gram measures of protein. Furthermore, the system is developed for the rating of different proteins or discrepancies among the same protein { Cho et al. , 2003 } .
The chief characteristics of the transeunt look systems are: simple in building of look vectors, time-efficient: generate merchandises in yearss, genetically stable and consistent, multiple processing, broad pertinence among assorted host cells { Wurm et al. , 1999 } . As ‘process is merchandise ‘ is a cardinal factor in the biopharmaceutical industry, the demand of better techniques for DNA transportation is indispensable for the large-scale recombinant protein production in mammalian cells. Many engineered viral vectors such as adenoviruses, alphaviruses, baculoviruses, lentiviruses etc. are used for developing big sums of recombinant proteins but they conjoin drawbacks such as restricted tropism, low transfection capacity and high cost of production { Pham et al. , 2006 } .Non-viral bringing systems have been developed such as Ca phosphate, electroporation, lipid reagents and reported higher transfection capableness with disciple every bit good as suspension adapted mammalian cells { Wurm,1999 } . Different cell lines used for non-viral mediated transfection are COS ( African green monkey kidney ) { Blasley et al.
, 1996 } , HEK-293 ( Human embryonic kidney ) { Jordan et al. , 1998 } , CHO ( Chinese Hamster Ovary ) { Preuss, 2000 } , BHK ( Baby hamster kidney ) { Wurm, 1999 } . Non-viral bringing systems has made important advancement in developing better look vectors, pick of cell lines, civilization medium, transfection parametric quantities and procedure scale-up { Pham,2006 } .
Non-viral Gene Delivery/Transfection
The cardinal factors of an ideal cistron bringing system are: protect Deoxyribonucleic acid from nucleases, translocate DNA into the karyon of the mark cell and have no inauspicious consequence { Gao et al. , 2007 } .
Viral bringing systems that have been developed for transportation of bare DNA into the mammalian cells are non really effectual as compared to non-viral systems as former by and large translocate plasmid DNA into cytol instead than karyons and therefore proved to be less effectual or really small cistron look detected { lungwitz et al. , 2005 } . Therefore vehicles with atomic translocation ( coupled with DNA and advance entry into the cell ) potency must be employed for large-scale production of the recombinant proteins.There are two types of non-viral cistron bringing systems carrier-free cistron bringing ( physical: needle injection, cistron gun, ultrasound, electroporation ) and man-made vector based cistron bringing ( chemical: Man-made polymers, lipoids and proteins ) { Gao et al. , 2007 } . Cationic polymers, cationic lipoids, atomic proteins ( histones ) have shown to be effectual vehicles { Colosimo et al. , 2000 } { Haberland et al. , 2005 } but they are extremely expensive for a large-scale production.
Zauner et Al. has compared transfection capacity of two liposomal composites known as Lipofectamine against poly ( l-lysine ) ( PLL ) and found polycations have greater transfection capableness { Godbey et al. , 2001 } .Electroporation of plasmid DNA is besides practised but this system requires development of more preciseness as it causes cell decease by electrical discharge { Pham et al. , 2006 } .
Calcium phosphate DNA co-precipitation and polyethyleneimine ( PEI ) Deoxyribonucleic acid composites are the most efficient vehicle systems because they are cost-efficient and could be used for large-scale production of both adapted and suspension cell lines, besides they transfer Deoxyribonucleic acid by organizing composites which are taken up by the cells through endocytosis { Wurm et al.,1999 } .Table 1: Gene Delivery barriers and multicomponent design of Non-viral vectors { Khanna et al.
, 2007 }Calciferol: nai-tbl3.jpg
Calcium-phosphate ( CaPi ) Precipitation
Graham and Van der Eb developed calcium-phosphate DNA co-precipitation method in 1973 and since so it has been the most efficient transfection system in mammalian cells { Pham et al. , 2006 } .
Precipitation of calcium-phosphate is formed by nucleation or atom growing and depends on the impregnation, at low supersaturation the atom growing exaggerates but due to undersaturation the atom redissolves { Jordan et al. , 2004 } . Therefore Ca phosphate method is deft and dynamic in nature, causes the alteration in features of the atom by the uninterrupted action of Deoxyribonucleic acid on the precipitate { Coonrod et al. , 1997 } .CaPi transfection by and large involves a two-step protocol:Adding Deoxyribonucleic acid to calcium chloride solutionFurthermore, blending it with phosphate to organize Ca phosphate DNA precipitatesAdding composites to the civilization medium { Graham et al. , 1973 } .
The CaPi DNA composites enter the mark cells by the procedure of phagocytosis and so high Ca concentration causes the rupture of the cysts. It has been found that plasmid DNA translocate into the karyon within 1h of transfection { Coonrod et al. , 1997 } . Besides work has been done to demo transportation of 1000s of plasmid DNA into single cell utilizing Ca phosphate transfection. The concentration of the Ca phosphate is the chief factor that effects its hasty formation but other parametric quantities such as temperature, pH and DNA concentration are besides of import { Jordan et al.
, 1996 } . The tabular array below describes the critical parametric quantities that affect CaPi precipitate formation.Table 2: Critical factors set uping Ca phosphate precipitate { Jordan et al. , 2004 } .
Polyethylenimine ( PEI )
In contrast of other non-viral vectors, polymers have been widely used for the transfection of mammalian cells due to their features of easy readying, purification and chemical stableness { lungwitz et al. , 2005 } . PEI is extremely polycationic man-made polymer that can be derived by condensation of 2-ethyl-2-oxazoline monomers or azridine and is available in additive or bifurcate constructions severally.
PEI is extensively used for its features of low cost, possible for operation at graduated table, minimum cytotoxity and efficient transgene look.The bifurcate PEI can be used for plasmid DNA bringing every bit good as for RNA or oligonucleotides and integral ribozymes { Aigner et al. , 2002 } . The efficiency of bPEI- derived vectors depends upon their features of molecular weight, grade of ramification, the cationic charge denseness, buffer capacity, polyplex capacity of DNA binding, content, size and absence or presence serum while transfection { lungwitz et al. , 2005 } .The bPEI with higher molecular weight shows higher transfection capableness along with stable complex formation as compared to the bPEI with lower molecular weight { Godbey et al. , 1999 } .
With the addition in N/P ratios increases the net positive charges on the composites therefore help in cell interactions and atomic uptake { Oh et al. , 2002 } .Figure: 1 Synthesis of branched polyethylenimine by condensation of azridine in aqueous solution { Harpe et al. , 2000 } .
Linear PEI ( lPEI ) distinguishes itself from the branched PEI from the atomic uptake transfection efficiency and toxicity. It is synthesized by polymerisation of either unsubstituted or two substituted 2 oxazolines with acid or base hydrolysis of N-substituted polymer ( Fig2 ) { Brissault et al. , 2003 } . It has been observed with increasing measures of amino groups in the copolymer besides effects the buffer capacity and transfection efficiency of the lPEI composites, besides big atoms of the lPEI shows higher interaction with cell surfaces { lungwitz et al. , 2005 } . In comparing with bPEI, lPEI showed improved cell viability, promote atomic localisation and increased transfection efficiency { Zou et al. , 2000 } .Fig2: Synthesis of additive polyethylenimine { Brissault et al.
, 2003 } .The intracellular trafficking tracts have revealed the apprehension of the PEI- mediated cistron transportation. Plasmid DNA packed with PEI is transferred to the cell by receptor-mediated endocytosis and therefore can be used for labelling polyplexes with aiming medieties ( Fig3 ) . Cationic polyplexes interact with the negatively charged membrane glycoproteins, proteoglycans, and sulphated proteoglycans and get down cell internalisation via adsorbent or fluid-phase endocytosis { lungwitz et al. , 2005 } . The responsiveness between cell and polyplex could be enhanced by increasing the net positive charge on the surface of the polyplex ( alteration in concentration or incubation clip ) { Bieber et al.
, 2002 } . The transfection of cell by PEI-DNA polyplexes starts within 3h after their incubation to the medium { Kristensen et al. , 2001 } .The PEI-DNA composites win through the endolysomal tract and escapes expeditiously endosomes/lysosomes by the ‘Proton Sponge ‘ consequence by which protonable amino N nowadays at every 3rd atom of PEI buffer the endosomal environment and hence apprehension acidification and merger with the lysosomes and finally swell the cysts to let go of plasmid DNA into the cytol { Akinc et al.
, 2005 } . PEI-DNA composites have shown high cistron look resulted in apprehension of the fact that it by and large transfers plasmid Deoxyribonucleic acid from cytol to the nucleus { Pollard et al. , 1998 } . The rate of PEI-mediated transfection and transgene look depends upon the cell-type and cell civilization medium { Kristensen et al. , 2001 } .
Experiments have been performed to understand the mechanism of protection of DNA by PEI and it has been stated that it protects Deoxyribonucleic acid from debasement by DNase1 and DNase 2, protecting the Deoxyribonucleic acid from debasement would take down cistron look degree so protecting it while bringing is an of import parametric quantity { Godbey et al. , 2001 } .ewaFig3: Conventional representation of the cistron transportation utilizing ligand-decorated non-viral vectors { Kircheis et al. , 2001 } .
Transient ( Non-Viral ) Transfection of Mammalian Cells
Transient cistron look is a rapid method of direct transition of recombinant DNA into recombinant protein.
It has the undermentioned advantages over stable cistron look:Expression over few days/weeksTransfect in many transcripts of Deoxyribonucleic acidTranscription without integrating into host genomeNo subculturingNo choice for care of look vectorQuickly generates protein for protein checks, word picture etc.Rapid for mg to gram sumsCost effectualFor big scale transient production of recombinant protein in mammalian cells, the cardinal factors that should be taken into history are:The cell lineThe look vectorThe plasmid DNA qualityThe transfection vehicleThe civilization medium { Durocher et al. , 2002 } .Large graduated table recombinant protein production depends upon the transfection capacity and for the development of such system requires the plasmid DNA showing newsman proteins like secreted alkaline phosphatase ( SEAP ) and green fluorescent protein ( GFP ) { Pham et al.
, 2006 } .
Mammalian cell lines
For the efficient output of recombinant protein the cell line should be easy transfected. Many different cell lines have been used in past such as COS and CV1 derived from African green monkey for the little graduated table transient production. The chief drawback with these cells is that they show less productiveness in contrast with other cell lines. Nowadays, the cell lines that are industrially accepted for the production of recombinant protein are Chinese hamster ovary ( CHO ) , mouse myeloma, babe hamster kidney and human embryologic kidney 293 ( HEK293 ) .
Chinese Hamster Ovary ( CHO )
CHO cell line is widely used for the production of recombinant proteins utilizing stable cistron look but less work has been done with transeunt transfection.
Initial work with these cell lines utilizing CaPi-mediated transient transfection has non shown hearty consequences as but ulterior work with PEI- mediated transeunt transfection has given improved productiveness of upto 10mg/L for antibodies { Wurm et al.,1999 } . CaPi requires serum and osmotic daze for the production of high degrees of protein but show low degree of cistron bringing with suspension-adapted CHO cell lines { } .
The highest transeunt outputs of 80-100mg/L have been reported by transfecting CHO cells with additive PEI ( 25 kDa ) { Wulhfard et al. , 2008, 2010 } { Ye et al. , 2009 } . In 2004 Derouazi et Al. has worked on the serum free suspension adapted CHO cells and scaled up the procedure in 20L moved bioreactor for the production of recombinant protein utilizing PEI-mediated cistron bringing ( Fig.4 ) { Derouazi et al.
, 2004 } .Recently Rajendra et Al. has reported volumetric productiveness of recombinant antibody of upto 300mg/L by optimising the transeunt transfection parametric quantities in suspension-adapted CHO cells { Rajendra et al. , 2011 } .Fig4: Transfection of CHO cells in 3L and 20L bioreactor transfected at a Deoxyribonucleic acid: PEI ratio of 1:2 { Derouazi et al. , 2004 } .
Human Embryonic Kidney 293
Human embryologic kidney 293 cell line is derived from transformed HEK cells with DNA of adenovirus 5 { Graham et al. , 1977 } .
These cells have been widely used for the production of recombinant protein utilizing transeunt cistron look as they are extremely susceptible to most cistron bringing systems and can turn in serum free suspension adapted medium. There are two different types of HEK 293 cells viz. 293E ( express EBNA1 ) and 293T ( express SV40 big T-Ag ) but both support episomal reproduction. The SV40 ori Tag system in 293 T cells does non advance high degrees of transient production in these cell lines because of coincident happening of two procedure of cistron reproduction and look { Pham et al. , 2006 } . In contrast 293E cell lines are widely developed for big scale transient production utilizing EBV oriP plasmids { Pham et al.
, 2003, 2005 } . HKB11 is a new intercrossed cell line developed by blending 293 with Burkitt ‘s lymphoma cell line and reported 20 fold high transient cistron look utilizing oriP/EBNA1 based plasmid { Cho et al. , 2003 } .CaPi and PEI are extensively used cistron bringing systems in HEK-293 cell line and former has been utilized for scaled up to 100L in bioreactors { Baldi et al.
, 2005 } . Durocher et Al. used theoretical account proteins such as SEAP and GFP to plan look vector and proctor transfection efficiency in 293E cell utilizing 25kDa PEI ( Fig5 ) { Durocher et al. , 2002 } .Fig 5: Transfection of different HEK293 cell lines with Deoxyribonucleic acid: PEI ratio at 1:2 for SEAP look and transfection efficiency { Durocher, 2002 } .
Expression Vectors
They play a important function for recovering high outputs of r-proteins utilizing transeunt cistron look. CMV booster is the most exploited among the mammalian cell lines, Xia et Al.
have compared different homo, rat and mouse CMV boosters and reported highest look with the hCMV in 293E and CHO cells { Xia et al. , 2006 } . Other booster showing elongation factor 1? ( EF1 ? ) is besides used in the CHO and HEK293 cell lines and has been reported more active than mCMV/EF1 booster in Cho cells { Derouazi et al. , 2004 } . A intercrossed metallothionein ( MT ) booster has been shown to be more active than intron-less boosters ( hCMV and SV40 ) in CHO cells { Pham et al. , 2006 } .
EBV based vectors show betterment in big graduated table TGE by adhering EBNA1 to the part of oriP, besides Cachianes et Al. reported 10 crease higher look in 293 cells due to presence of oriP { Cachianes et al. , 1993 } .For the production of heterodimeric recombinant proteins such as antibodies it is necessary to cotransfect two vectors ( coding separate polypeptides ) to obtain complete antibody molecule, therefore Baldi et Al. performed experiments utilizing different ratios of two vectors pEAK-LH39 and PEAK-LH41 in suspension HEK 293 cells with the Ca-Pi transfection method ( Fig 6 ) { Baldi et al. , 2005 } .Fig 6: Cotransfection of HEK293 cells with heavy ( H ) and light ( L ) concatenation coding DNA vectors utilizing CaPi method, the highest antibody look was observed with equal H/L ratio { Baldi et al.
, 2005 } .
Plasmid DNA Amount
As reported a 45 L graduated table transfection by and large requires 45 or 56 milligram of plasmid DNA for CaPi or PEI methods, therefore plasmid DNA could be generated by utilizing 2-4 L of E.coli civilization medium { Pham et al. , 2006 } . Extraction of plasmid Deoxyribonucleic acid could be done by alkalic lysis protocol postulated by Birnboim and Dolly { Birboin et al.
, 1979 } . Purification of supercoiled Deoxyribonucleic acid is by and large done by size exclusion and ion exchange chromatography.Fig 7: A.
Change in transfection efficiency C. Antibody look degree with changing sum of DNA as PEI was kept changeless in CHO cells { Rajendra et al. , 2011 } .
Transfection ( Non-viral ) Vehicles
CaPi
Calcium phosphate precipitation is dynamic and reversible in nature and therefore depends upon several critical parametric quantities such as cell denseness, concentration of DNA, Ca phosphate, pH of the phosphate solution and clip of medium dilution { Jordan et al. , 2004 } . The chief drawback with CaPi-mediated transfection is its dependance on serum and demand of media replacing before transfection ; due to these factors it is hard to transfect suspension adapted CHO cells { Derouazi et al. , 2004 } .Another attack similar to CaPi precipitation has been developed called ‘Calfection ‘ but it still requires serum for the efficient production of r-proteins. Jordan et Al. showed the consequence of phosphate concentration in the precipitation mixture and reported that higher concentration of phosphate has a negative consequence on transfection efficiency ( Fig 8 ) { Jordan et al. , 2004 } .
Fig8: Consequence of phosphate concentration on turbidness, DNA binding and recombinant protein look degree for adherent CHO and adherent HEK cells { Jordan et al. , 2004 } .
Pei
PEI-DNA polyplexes are the most efficient non-viral cistron bringing system widely used among assorted mammalian cell lines for the production of high degree of protein look. It performs scaleable transfection procedure and therefore provides recombinant protein outputs in equality to CaPi ( Table-3 ) { Baldi et al. , 2005 } .Table-3: Comparison between PEI and CaPi-mediated transfection under serum-free conditions for the production of recombinant protein { Baldi et al.
, 2005 } .HEK293 cells transfected with PEI-mediated transient system showed that 25kDa branched PEI is more effectual than remainder of the molecular weights of PEI, besides 25L PEI is more effectual than 25B PEI for the transfection of suspension turning 293E cells { Durocher, 2002 } . The highest outputs have been reported by transfection of HEK-293 cells with 25 kDa additive PEI at a cell denseness of 20 ten 106 cell/ml by subsequently add-on of PEI and DNA to the medium incorporating valproic acid ( VPA ) { Backliwal et al. , 2008c } .
Carpentier et Al. has demonstrated the consequence of sum of polyplex on the feasible cell count, mRNA degree and protein production in HEK293-EBNA1 cells transfected with 100 % , 150 % and 200 % polyplex concentration and found that mRNA degree was increased by utilizing standard polyplex sums ( Fig9 ) { Carpentier et al. , 2007 } .Fig 9: Consequence of polyplex sum on feasible cell count, mRNA degree and protein production in HEK293-EBNA1 cells { Carpentier et al. , 2007 } .
Culture medium
Serum-free media is extremely appreciated in the biopharmaceutical industry because it eradicates the possible viruses and therefore makes the downstream processing of recombinant protein easy. But serum-free media has shown low transfection efficiency as compared to serum- incorporating media { Geisse et al. , 2005 } .
Negatively charged polyplexes interact with proteoglycans of membrane, as heparin nowadays in serum free media causes the neutralisation of these polyplexes and consequence in low transfection efficiency { Mislick et al. , 1996 } .Durocher et Al. demonstrated the consequence of serum on transfection efficiency ( GFP ) and recombinant protein look degree ( SEAP ) , transfection of suspension turning human2293- EBNA1 cells was done by both additive and branched PEI { Durocher et al. , 2002 } .
The cells were added to the transfection mixture incorporating 1 % serum and showed a 4-5 fold addition in protein look degree as compared to when added to the serum-free media ; besides with addition in concentration of serum from 1 % to 5 % it farther showed betterment in transfection efficiency and protein production { Durocher et al. , 2002 } . In contrast when cells where added to the serum free medium they showed 50 % less productiveness as compared to the serum incorporating media.Fig10: Consequence of Serum on transgene look { Durocher et al. , 2002 } .
Decision
Transient cistron look is a rapid method of direct transition of recombinant DNA into recombinant protein. Large graduated table recombinant protein production depends upon the transfection capacity and for the development of such system requires the plasmid DNA showing newsman proteins like secreted alkaline phosphatase ( SEAP ) and green fluorescent protein ( GFP ) { Pham et al.,2006 } .Non-viral mediated transfection has resulted in production of mgs to gram measures of recombinant proteins. Calcium phosphate precipitation ( CaPi ) and polyethyleimine ( PEI ) are the most extensively used non-viral cistron bringing systems in mammalian cells. The CaPi DNA composites enter the mark cells by the procedure of phagocytosis and so high Ca concentration causes the rupture of the cysts whereas PEI-DNA composites win through the endolysomal tract and escapes expeditiously endosomes/lysosomes by the ‘Proton Sponge ‘ consequence.
CaPi and PEI are extensively used cistron bringing systems in HEK-293 cell line and former has been utilized for scaled up to 100L in bioreactors { Baldi et al. , 2005 } . Critical factors of transfection vehicles such as pH, phosphate concentration, DNA concentration, besides affect the overall recombinant protein productiveness. Both CaPi and PEI perform scaleable transfection procedure and therefore supply equal measures of recombinant protein { Baldi et al. , 2005 } .Effect of serum is besides important in big scale transient production of recombinant protein and straight affects the transfection efficiency.
