How To Immortalize Primary Cells

Primary Cells Can Not Expand Several Rounds Of Proliferation, Which Is Called Hayflick Limit, Because Each Round Of Proliferation Will Shorten Telomeres. When The Telomere Reaches The Critical Shortening Length, DNA Damage Is Triggered, Leading To Cell Aging. If You Try To Cultivate A Rare Primary Cell Group, Be Careful That It Will Eventually Die, You Can Manipulate The Cell To Bypass The Aging Process And Become Immortal. The Immortalization Of The Primary Cell Is Such A Magical Thing& Nbsp; The Best Example Of Spontaneous Immortalized Cells Is Cancer Cells, Which May Have Undergone Genetic Changes To Resist Aging And Begin To "live Forever". In Fact, Many Cancer Cell Lines Have No Such Change. In Fact, George Gey, A Scientist, Created The First Immortal And Arguably The Most Famous Cell Line: HeLa Cells. He Accidentally Found That There Were Highly Developed Uterine Cancer Cells In The Tumor After Henrietta's Surgery. Before That, He Tested Hundreds Of Cancer Cells To Obtain Immortality, But Failed& Nbsp; Many Viral Genes Affect The Cell Cycle, So Aging Can Be Overcome By Eliminating The Biological Brake Of Proliferation Control. Many Of Them Are Tumor Suppressor Genes, Because They Need To Inhibit The Occurrence Of Tumors. The Most Common Is The Overexpression Of Simian Virus Large T Antigen (SV-40), Which Inhibits The Rb Gene And P53 Gene Of Cells, Both Of Which Are Key Controllers Of Cell Growth Cycle. A Famous Example Of SV40 Immortalized Cell Lines Is HEK293T, Also Known As 293T Cells. This Is A Cell Line Widely Used To Express Virus Particles And Cell Analysis, Because They Are Easy To Transfect. Other Viral Genes Include Genes From Human Papillomavirus (HPV), Such As E6 And E7, Which Also Target Rb And P53 Genes, Such As Human Bronchial Epithelial Cell HBE-E6E7& Nbsp; The Most Famous Immortal Gene Is Telomerase (hTERT). Telomerase Is A Ribonucleoprotein, Which Can Prolong The DNA Sequence Of Telomeres, Thus Slowing Down The Aging Process And Enabling Cells To Undergo Unlimited Cell Division. In Fact, Telomerase Has Recently Been Considered As A Potential Mechanism To Reverse Aging. The Problem With Using Telomerase To Reverse Aging Is That Increased Telomerase Expression Can Also Induce Tumor Growth. In Fact, Many Genes Used To Create Immortalized Cell Lines, Such As HTERT And SV40, Can Induce Tumor Formation. Therefore, It Is Not Surprising That HTERT Is Often Overexpressed In Human Tumors, Which Endows Cancer With A Key Feature: Unrestricted Proliferation. This Is Exactly The Purpose That We Use It To Immortalize The Primary Cell Line& Nbsp; In Some Types Of Cells, Only One Immortalization Method May Produce A Small Number Of Immortalized Cells. Therefore, According To Your Cell Line, Combine The Inhibition Of Tumor Suppressors (such As The Above Cell Cycle Inhibitors) With The Expression Of HTERT To Increase The Possibility Of Cell Immortalization. If You Want To Immortalize And Transform Your Cell Lines (even If They Are Like Tumorigenic Cells), It Is Also Recommended To Use This Dual Method, Because There Is Evidence That Some Cell Lines Cannot Be Transformed Effectively Without Immortalization& Nbsp; How To Introduce Immortality Into Many Immortal Sources Above The Primary Cell Line Is Based On The Genetic Manipulation Of The Primary Cell. This Requires The Introduction Of Foreign DNA Into The Cell. Because Many Primary Cells Are Difficult To Transfect, The Simplest And Most Effective Way To Introduce Gene Changes Into Primary Cells Is To Infect Them Through Viruses& Nbsp; The Most Popular Method Is To Copy Defective Lentiviruses, Because They Are Safer Than Adenoviruses. Adenoviruses Have The Ability To Re Infect Cells, So They Contain Live Viruses For A Longer Time. Retroviruses Can Also Be Used To Transfect Cells, However, They Can Only Infect Actively Dividing Cells, Thereby Reducing The Number Of Cells That May Be Transfected By Viruses& Nbsp;& Nbsp; Warning: Immortality Of Primary Cells May Not Be The Best Way& Nbsp; By Introducing Genetic Changes Into Cells, You May Be Profoundly Altering The Phenotype Of Cell Lines. Although This Does Make Your Cell Line More Useful In Some Aspects: It May Make Your Cells More Homogenous, So That You Can Easily Duplicate The Results. You Can Create A Large Number Of Cells For Future Use, And They May Be Easier To Experiment, Using The Primary Cell Line Still Has Many Advantages. The Primary Cells Can Maintain The Metabolism Of Normal Cells, Maintain The Physiological Characteristics Of Normal Cells, And Have The Unity And Repeatability Of The Experiment, However, With The Immortal Transfection Of Primary Cells, The Cell Population And Cell Mechanism Change, Which May Confuse Your Experiment And Lead To Uncertain Or Wrong Results. In Addition, There Are Still Many Debates About How Immortalized Cells Accurately Simulate Real Tissues& Nbsp; Although Controversial, SV40 Is Unlikely To Infect Humans, Although Its Mechanism Of Action: P53 And Rb Mutations May Be Very Common Mutations In Human Tumors. Therefore, It Is Important To Determine The Best Genetic Manipulation To Use Your Immortalized Cell Type& Nbsp; Although Immortalized Cells Are Very Powerful In Experimental Research, They Must Accept Some Warnings In Use. Like Any Experimental Model, Immortalized Cells Are Just Models Of The Cell System You Expect& Nbsp; The Following Is The List Of Spot Immortalized Cell Lines Commonly Used By Shanghai Qida Biotech: Article Reference: ATCC: HEK293T Cells (ATCC CRL-3216) Garbe  Et Al  (1999)  Viral Oncogenes Accelerate Conversion To Immortality Of Cultured Conditionally Immortal Human Mammary Epithelial Cells.& Nbsp; Oncogene.& Nbsp; 18(13): 2169  Callaway, E (2010) Nature News:  Telomerase Reverses Ageing Process.Hanahan & Weinberg (2011)  Hallmarks Of Cancer: The Next Generation.& Nbsp; Cell.& Nbsp; 144(5): 646.  Zhu  Et Al  (1991)  The Ability Of Simian Virus 40 Large T Antigen To Immortalize Primary Mouse Embryo Fibroblasts Cosegregates With Its Ability To Bind To P53.  Journal Of Virology.& Nbsp; 65(12): 6872.  Redell (1999)  The Role Of Senescence And Immortalization In Carcinogenesis.& Nbsp; Carcinogenesis.& Nbsp; 21(3): 477.