Cloning and Selection
Cloning
• Why Do We Need To Clone?
– To Isolate Cells With Specialized Properties
– Unspecialized Cells Tend To Dominate
– Cells Of Wrong Lineage Tend To Dominate
Isolation, Cloning Of
Specialized Cells
Overgrowth Of
Unspecialized Cells
Cloning
• Cloning Is Relatively Easy For Continuous Cell
Lines
• Difficult In Primary Cultures
• Nevertheless Possible
• A Serious Limitation Is Senescence
• Cloning Of Attached Cells Carried Out In
– Petri dishes
– Multiwell Plates
– Flasks
• Not Hard To Distinguish Individual Colonies
Cloning
• Cloning In Suspension
– Accomplished By Seeding Cells In A Gel (agar)
– Viscous Solution (Methocel) With Agar Underlay
• Viscous Matrix Ensures That Daughter Cells Remain In
Colony
• Hematopoietic Cells Usually Clone In Suspension
• However Most Normal Cells Typically Require
Adherence
• Suspension Cloning Is Used As Evidence For
Transformation
Dilution Cloning
• Dilution Cloning Was First Introduced By Puck
and Marcus, 1955
• It Is The Most Widely Used Technique
• Based On Observation That Cells Diluted Below
Certain Density Form Discrete Colonies
Dilution Cloning Protocol
• Trypsinize CHO (Chinese Hamster Ovarian) Cells,
Ensure Single Suspension
• When Detachment Is Observed Terminate Reaction With
5 mL Medium/FBS
• Count Cells And Dilute To 1x105 cells/mL
• Dilute To 10 cells/mL
– Ex. Take 200 L dilute to 20 mL (1x103 cells/mL)
– Repeat above dilution (1x101 cells/mL)
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Culture 0.1 mL in 96 well Plates (~1 cell/well)
Wait For A Week, Hopefully Clones Will Be Visible
If Not, Wait For An Additional Week
If Doing This For First Time, Use10, 20, 50, 100, 200
and 2,000 cells/mL To Determine Plating Efficiency
Number Of Cells
Clonal Cell Yield
109
106
20
30
Number Of Doublings
Plating Efficiency
• Low Density Plating Results In Low Survival Rate
• For Normal Cells Plating Efficiency Drops To
0.5%-5%
• Reasons For Low Plating Efficiency
– Loss By Leakage
– Cell Derived Diffusible Factors Too Dilute
• Capillary Technique Overcomes The Above
Limitations
– Confines Of Capillary Tube Allow For Locally
Enriched Environment
• Improved Media In Conjunction With Feeder
Cells Increase Plating Efficiency
Improving Clonal Growth
• Select Rich Medium
– Ex. Ham’s F12
• Hormones
– Insulin 1x10-10 IU/mL
– Dexamethasone 1x10-5 M for glia, myoblasts,fibroblasts
• Substrate Molecules
– Polylysine 1mg/mL Plate Coating, wash with PBS to remove
remaining
– Fibronectin 5g/mL in medium
• Conditioned Medium
– Medium used to grow other cells and added to regular
medium (care must be taken to avoid cross-contamination)
Improving Clonal Growth
• Feeder Cells
– Mimic high cell concentration
– Must be growth-arrested (mitomycin C or
irradiation)
– May provide nutrients, growth factors, matrix
• Feeder Cells Eventually Die
• Ex Of Feeder Cells
– 3T3, MRC-5 and STO cells
• Sensitivity To Irradiation/mitomycin C Varies
– Trial run is recommended
Cloning In Suspension
• Hematopoietic Cells Are Cloned In Suspension
• Colony Is Held Together By Viscous Medium
– Agar
– Methocel+Agar overlay
• Methocel Offers Advantages
– No impurities
– Easier to handle
• Colonies Form At Interface Between Methocel and
Agar Overlay
Methocel Protocol
• Prepare 0.6 % Agar Underlay
– 2x Medium with 40% FBS
– 1.2% Agar (UPW+1.2g agar)
– Add 1 mL to dishes, cover base, let set at R.T
• Dilute 0.8% Methocel with 2x Medium, Keep On Ice
• Prepare Cell Dilutions (1x105/mL, 3.3x104/mL, 1.1x104/mL,
3.7x103/mL)
• Add 40 L of each Dilution To Labeled Tubes + 4 mL Of 0.8%
Methocel, Vortex (Final Concentrations: 1,000;330;110;37 per dish)
• Use Syringe To Add 1 mL Of Each Dilution To Dishes
• Incubate In Humid Incubator Until Colonies Form
Isolation Of Clones (Adherent)
• Adherent Cells In Multi-well Plates Are
Trypsinized
• If In Petri Dishes No Physical Barrier Exists
Between Colonies
– Rings (ceramic, steel, plastic) are used
• Irradiation Can Also Be Used (30 Gy)
– Clone of interest is shielded with lead disk
• Clones Grown On Small Or Fragments Of Cover
Slips Are Physically Removed To New
Environment
Isolation Of Clones (Suspension)
• Isolation Is Easy But Requires Dissection
Microscope
• See Next Slide