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 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

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

• 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)

• 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

Dilution Cloning Protocol

Clonal Cell Yield

20

106

Number Of Doublings

Num

ber

Of

Cel

ls

109

30

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 5g/mL in medium

• Conditioned Medium– Medium used to grow other cells and added to regular medium (care

must be taken to avoid cross-contamination)

• 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

Improving Clonal Growth

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

• 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

Methocel Protocol

• 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 (Adherent)

• Isolation Is Easy But Requires Dissection Microscope

• See Next Slide

Isolation Of Clones (Suspension)