Physical method of transformation

Physical method of transformation- Microprojectile gun Bombardment, Electroporation and Microinjection

1. PHYSICAL METHOD OF TRANSFORMATION
Dr.Manikandan Kathirvel M.Sc., Ph.D., (NET)
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
(Reaccredited with "A" Grade by NAAC)
Affiliated to BengaluruNorthUniversity,
K. Narayanapura, Kothanur(PO)
Bengaluru 560077

2. PHYSICAL METHOD OF TRANSFORMATION:
Due to amphipathic nature of the phospholipidbilayerof the plasma membrane, polar
molecules such as DNA and protein are unable to freely pass through the membrane.
Various physical or mechanical methods are used in gene transfer in overcoming this
barrier.
They are:
1. Electroporation
2. Microinjection
3. Particle Bombardment
4. Sonoporation
5. Laser induced
6. Bead Transfection

3. I).ELECTROPORATION:
ThismethodwasfirstdemonstratedbyWongandNeumannin1982tostudygenetransfer
inmousecells.
Electroporationwasintroducedinthe1960sandcomprisestheapplicationofcontrolled
electricfieldstofacilitatecellpermeabilization.Thesuccessofinvitrodeliveryby
electroporationhasledtothedevelopmentofinvivoapplications.Thefirstinvitroandin
vivoattemptstouseelectroporationingenetransferweredemonstratedin1982and1991,
respectively.
Electroporationisaphysicaltransfectionmethodthatusesanelectricalpulsethat
temporarilydisturbsthephospholipidbilayerandcreatetemporaryporesincell
membranesthroughwhichsubstanceslikenucleicacidscanpassintocells.
Itisnowawidelyusedmethodfortheintroductionoftransgeneeitherstablyortransiently
intobacterial,fungal,plantandanimalcells.
Itisahighlyefficientstrategyfortheintroductionofforeignnucleicacidsintomanycell
types,includingbacteriaandmammaliancells.

4. Principle:
Electroporation is based on a simple process.
Host cells and selected molecules are suspended in a conductive solution, and an
electrical circuit is closed around the mixture.
Conditions: varying conditions of Voltage (V), resistance(Ω) and capacitor (µF) 25
Anelectricalpulseatanoptimizedvoltageandonlylastingafewmicrosecondstoa
millisecondisdischargedthroughthecellsuspension.
Thisdisturbsthephospholipidbilayerofthemembraneandresultsintheformationof
temporarypores.
Theelectricpotentialacrossthecellmembranesimultaneouslyrisestoallowcharged
moleculeslikeDNAtobedrivenacrossthemembranethroughtheporesinamannersimilar
toelectrophoresis.

5. Electroporation buffers
Choice of electroporation buffer depends on the cells being used in the experiment.
The following buffers (stored at 4°C) can be used:
1.Phosphate Buffered Saline (PBS) withoutCa
++
or Mg
++
2.HEPES-buffered saline
3.Tissue culture mediumwithoutFCS
4.Phosphate-buffered sucrose: 272 mMsucrose/7 mMK
2HPO
4(adjusted to pH 7.4
with phosphoric acid)/1 mMMgCl
2
5.0.4 M mannitolin PBS
6.5 mMCaCl
2 in PBS
7.0.5 M sucrose
Electroporation Cuvettes: Forelectroporationofmicroorganisms, 0.1 and 0.2 cm gap
cuvettes are most often used.
Electroporationof E. coliis generally carried out:
•at avoltageof 1.8 kV when electroporatingcells in 0.1 cm cuvettesand
•at avoltageof 2.5 kV when electroporatingcells in 0.2 cm cuvettes.

6. Procedure:
1.Apopulationoftargetcellsistakenandresuspendedthecellswiththeelectroporation
buffer.Theelectroporationbufferprotectscellsuspensionfrommechanicaldamage.
2.Alongwithit,targetDNAorplasmidDNAtakenandmixedinthecuvette.Thenitis
placedinanelectroporator.
3.Theelectroporationcuvettewithtwoaluminiumelectrodes,carryingthetargetcell
suspensionmixedwithplasmidortargetgene,isfittedintheelectroporationmachine
ordeviceknownaselectroporator.
4.Whenthedeviceisconnectedwiththepowersource,setwithvoltage(1200-2500V),
resistance(Ohm-Ω)andconstantcapacitor(25or50µF)andwhenitisswitchedon,
thecircuitcompletes,andthepulsedisgenerated.

7. 5.An electrical pulse is passed through the cell suspension. This will create
temporary/transient pores in the cell plasma membrane.Charged molecules like DNA can
pass through pores into the cytoplasm of the cell, under the influence of current.
6.Note:The entire process completes within a second (in a microsecond or millisecond).The
success rate of the entire process depends on two variables;pulse lengthandfield
strength.
7.Immediately, the cells are suspended into nutrient media, to revive the cells for 1-2 hr at
37°C and the viability/transformed cells are checked by plating the electroporated cells
onto the media containing respective antibiotic or other markers.

8. Applications
Electroporationiswidelyusedinmanyareasofmolecularbiologyandinmedicalfield.
Someapplicationsofelectroporationinclude:
DNAtransfectionortransformation
ElectroporationismainlyusedinDNAtransfection/transformationwhichinvolves
introductionofforeignDNAintothehostcell(animal,bacterialorplantcell).
Directtransferofplasmidsbetweencells
Itinvolvestheincubationofbacterialcellscontainingaplasmidwithanotherstrainlacking
plasmidsbutcontainingsomeotherdesirablefeatures.Thevoltageofelectroporation
createspores,allowingthetransferofplasmidsfromonecelltoanother.Thistypeoftransfer
mayalsobeperformedbetweenspecies.Asaresult,alargenumberofplasmidsmaybe
growninrapidlydividingbacterialcoloniesandtransferredtoyeastcellsbyelectroporation.
Genetransfertoawiderangeoftissues
Electroporationcanbeperformedinvivoformoreefficientgenetransferinawiderangeof
tissueslikeskin,muscle,lung,kidney,liver,artery,brain,corneaetc.Itavoidsthevector-
specificimmune-responsesthatareachievedwithrecombinantviralvectorsandthusare
promisinginclinicalapplications.

9. Advantages
1.It is highly versatile and effective for nearly all cell types and species.
2.It is highly efficient method as majority of cells take in the target DNA molecule.
3.It can be performed at a small scale and only a small amount of DNA is required as
compared to other methods.

10. II).MICROINJECTION
DNAmicroinjectionwasfirstproposedbyDr.MarshallA.Barberintheearlyofnineteenth
century.
1.Thismethodiswidelyusedforgenetransfectioninmammals.
2.Thismethodhasbeensuccessfullyappliedtogeneticallymodifiedanimalsincluding
mice,fish,rats,rabbitsandmanylargedomesticanimalssuchascattle,sheepandpigs.
3.ItinvolvesdeliveryofforeignDNAintoalivingcell(e.g.acell,egg,oocyte,embryosof
animals)throughafineglassmicropipette.TheintroducedDNAmayleadtotheoveror
underexpressionofcertaingenes.
4.Itisusedtoidentifythecharacteristicfunctionofdominantgenes.

11. Procedure:
1.ThedeliveryofforeignDNAisdoneunderapowerfulmicroscopeusingaglass
micropipetteneedleswithveryfinetip(0.1to0.5μm)todirectlyinjectforeigngene
fragmentsintopronuclearembryosorculturedcells.
2.Cellstobemicroinjectedareplacedinacontainer.Aholdingpipetteisplacedinthe
fieldofviewofthemicroscopethatsucksandholdsatargetcellatthetip.
3.Thetipofmicropipetteisinjectedthroughthemembraneofthecelltodeliverthe
contentsoftheneedleintothecytoplasmandthentheemptyneedleistakenout.
Delivery of DNA into a cell through microinjection

12. Advantages
1.• No requirement of a marker gene.
2.• Introduction of the target gene directly into a single cell.
3.• Easy identification of transformed cells upon injection of dye along with the DNA.
4.• No requirement of selection of the transformed cells using antibiotic resistance or
herbicide resistance markers.
5.• It can be used for creating transgenic organisms, particularly mammals.
Disadvantages
1.Injection can cause damage that affects embryonic survival and can result in quite
high mortalities.
2.Only one cell is targeted per injection.

13. III.)PARTICLEBOMBARDMENTMETHOD
•ItwasinventedbyProf.JohnCSanford,EdWolfandNelsonAllenatCornellUniversity
(USA),andTedKleinofDuPont,between1983and1986developedtheoriginal
bombardmentconceptandcoinedtheterm“biolistics”(shortfor“biologicalballistics”)for
boththeprocessandthedevice.
•Theoriginaltargetwasonions(chosenfortheirlargecellsize)anditwasusedtodeliver
particlescoatedwithamarkergene.Genetictransformationwasthenprovenwhenthe
oniontissueexpressedthegene.
•Alsotermedasparticlebombardment,particlegun,shotgun,microprojectile
bombardmentandparticleacceleration.
•Itemployshigh-velocitymicroprojectilestodeliversubstancesintocellsandtissues.
Macrocarrier

14. Apparatus
1.Thebiolisticguninvolvestheprincipleofthepassageofheliumgasthroughthecylinder
witharrangeofvelocitiesrequiredforoptimaltransformationofvariouscelltypes.
2.Itconsistsofabombardmentchamberwhichisconnectedtoanoutletforvacuum
creation.
3.Thebombardmentchamberconsistsofaplasticrupturediskbelowwhichmacrocarrieris
loadedwithmicrocarriers.
4.ThesemicrocarriersconsistofgoldortungstenmicropelletscoatedwithDNAfor
transformation.
B
Macrocarrier

15. 5.TheapparatusisplacedinLaminarflowtomaintainsterileconditions.
6.Thetargetcells/tissueisplacedintheapparatusandastoppingscreenisplaced
betweenthetargetcellsandmicrocarrierassembly.
7.Thepassageofhighpressureheliumrupturestheplasticrupturediskpropellingthe
macrocarrierandmicrocarriers.
Working system of particle bombardment gun
8.ThestoppingscreenpreventsthepassageofmacroprojectilesbutallowstheDNA
coatedmicropelletstopassthroughitthereby,deliveringDNAintothetargetcells.
Macrocarrier

16. Procedure:
Step1:Thegenegunapparatusisreadytofire.Heliumfillsthechamberandpressure
buildsagainsttherupturedisk.
Step2:TheDNAtobetransformedintothecellsiscoatedontomicroscopicbeads
madeofeithertungstenorgold.Thecoatedbeadsarethenattachedtotheendofthe
plasticbullet(macrocarrier)andloadedintothefiringchamberofthegenegun.
Step3:Thepressureeventuallyreachesthepointwheretherupturediskbreaks,and
theresultingburstofheliumpropelsmacrocarrierandhitstheDNA/gold-coated
microcarrierintothestoppingscreen.
Step4:whenthemacrocarrierhitsthestoppingscreen,theDNA-coatedgoldparticles
arepropelledthroughthescreenandintothetargetcells.
Macrocarrier
The stopping screen prevents the passage of macro projectiles but
allows the DNA coated micro pellets to pass through it thereby,
delivering DNA into the target cells

17. Uses
1.•Thismethodiscommonlyemployedforgenetictransformationofplantsandmany
organisms.Thistechniquehasbeenusedsuccessfullytotransformsoyabean,cotton,
spruse,sugarcane,papaya,sunflower,rice,maize,wheatandtobacco.
2.•Theparticlegunhasalsobeenusedwithpollen,earlystageembroyoids,meristemsand
somaticembryos.
3.•Thismethodisapplicablefortheplantshavinglessregenerationcapacityandthose
whichfailtoshowsufficientresponsetoAgrobacterium-mediatedgenetransferinrice,
corn,wheat,chickpea,sorghumandpigeon-pea.
4.GenegunshavealsobeenusedtodeliverDNAvaccines.
5.Thedeliveryofplasmidsintoratneuronsthroughtheuseofagenegun,specificallyDRG
neurons,isalsousedasapharmacologicalprecursorinstudyingtheeffectsof
neurodegenerativediseasessuchasAlzheimer'sdisease.
6.Thegenegunhasbecomeacommontoolforlabelingsubsetsofcellsinculturedtissue.In
additiontobeingabletotransfectcellswithDNAplasmidscodingforfluorescentproteins,
thegeneguncanbeadaptedtodeliverawidevarietyofvitaldyestocells.
7.GenegunbombardmenthasalsobeenusedtotransformCaenorhabditiselegans,asan
alternativetomicroinjection.

18. Advantages
•SimpleandconvenientmethodinvolvingcoatingDNAorRNAontogoldmicrocarrier,
loadingsamplecartridges,pointingthenozzleandfiringthedevice.
•Noneedtoobtainprotoplastastheintactcellwallcanbepenetrated.
•Manipulationofgenomeofsub-cellularorganellescanbedone.
•Eliminatestheuseofpotentiallyharmfulvirusesortoxicchemicaltreatmentasgene
deliveryvehicle.
•ThisdeviceofferstoplaceDNAorRNAexactlywhereitisneededintoanyorganism.
•TechnicalsimplicityandRelativelyhightransformationefficiency.
Disadvantages
•ThetransformationefficiencymaybelowerthanAgrobacterium-mediated
transformation.
•Specializedequipmentisneeded.Moreoverthedeviceandconsumablesarecostly.
•Associatedcelldamagecanoccur.
•Thetargettissueshouldhaveregenerationcapacity.
•Randomintegrationisalsoaconcern.
•Chancesofmultiplecopyinsertionscouldcausegenesilencing.