Cloning and Selection Cloning Why Do We Need
- Slides: 20
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 m. L Medium/FBS • Count Cells And Dilute To 1 x 105 cells/m. L • Dilute To 10 cells/m. L – Ex. Take 200 L dilute to 20 m. L (1 x 103 cells/m. L) – Repeat above dilution (1 x 101 cells/m. L) • • Culture 0. 1 m. L 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, Use 10, 20, 50, 100, 200 and 2, 000 cells/m. L 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 F 12 • Hormones – Insulin 1 x 10 -10 IU/m. L – Dexamethasone 1 x 10 -5 M for glia, myoblasts, fibroblasts • Substrate Molecules – Polylysine 1 mg/m. L Plate Coating, wash with PBS to remove remaining – Fibronectin 5 g/m. L 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 – 3 T 3, 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 – 2 x Medium with 40% FBS – 1. 2% Agar (UPW+1. 2 g agar) – Add 1 m. L to dishes, cover base, let set at R. T • Dilute 0. 8% Methocel with 2 x Medium, Keep On Ice • Prepare Cell Dilutions (1 x 105/m. L, 3. 3 x 104/m. L, 1. 1 x 104/m. L, 3. 7 x 103/m. L) • Add 40 L of each Dilution To Labeled Tubes + 4 m. L Of 0. 8% Methocel, Vortex (Final Concentrations: 1, 000; 330; 110; 37 per dish) • Use Syringe To Add 1 m. L 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
- Pictures
- Two way selection and multiway selection in c
- Multiway selection
- Mass selection and pure line selection
- Scientific selection process in hrm
- Pros of cloning
- Identity cloning and concealment
- Cloning and sequencing explorer series
- Advantages and disadvantages of cloning
- Advantages and disadvantages of cloning
- Don't ask why why why
- Why do we need to study the nature and aims of business?
- Balancing selection vs stabilizing selection
- Similarities
- K selection r selection
- Natural selection vs artificial selection
- Difference between continuous and discontinuous variation
- What is stabilizing selection
- What is exponential growth in ecology
- Natural selection vs artificial selection
- Gene cloning process