1. Cloning and Selection

2. 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

4. 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

5. 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

6. 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

9. 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 1x10 5 cells/mL Dilute To 10 cells/mL –Ex. Take 200  L dilute to 20 mL (1x10 3 cells/mL) –Repeat above dilution (1x10 1 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

10. Clonal Cell Yield 20 10 6 Number Of Doublings Number Of Cells 10 9 30

11. 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

12. 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)

13. 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

15. 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

16. 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 (1x10 5 /mL, 3.3x10 4 /mL, 1.1x10 4 /mL, 3.7x10 3 /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

18. 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)

19. Isolation Is Easy But Requires Dissection Microscope See Next Slide Isolation Of Clones (Suspension)