How gene and cell work fits together

Think of a gene as a recipe. To learn what a recipe makes, you copy it carefully, put it in an envelope with a delivery address (a vector), deliver it to the kitchen (the cell), decide when cooking starts (an on/off switch), and taste the result (measure RNA or protein).

Typical customers: University and institute research groups, biotech R&D, cancer and infection biology labs

1. 1

   Copy the gene

   PCR makes millions of exact copies of the gene from DNA or cDNA, or you order the gene ready-made as synthetic DNA.

   • Takara Bio CloneAmp HiFi PCR Premix 639298
2. 2

   Put it into a vector (cloning)

   The gene copy is joined into a carrier DNA called a vector, which has an address label (promoter) and a selection marker. In-Fusion joins pieces by matching 15-letter overlaps, so no restriction sites or ligase are needed.

   • Takara Bio In-Fusion Snap Assembly Master Mix 638947
   • Takara Bio Stellar Competent Cells 636763
   • Microsynth Sanger sequencing
3. 3

   Add an on/off switch optional

   An inducible system keeps the gene silent until you add doxycycline (a harmless antibiotic) to the culture, then switches it on. Remove it and the gene goes quiet again.

   • Takara Bio Tet-One Inducible Expression System 634301
4. 4

   Deliver it into cells

   Easy cells (HEK293, HeLa, CHO) take up plasmid with a chemical carrier: transfection. Hard cells (primary cells, stem cells, T cells, neurons) need a disabled virus used purely as a delivery van: lentivirus or AAV. The virus cannot copy itself.

   • Takara Bio Xfect Transfection Reagent 631317
5. 5

   Grow the cells

   Cells need a medium (food), serum (growth factors) and clean handling. Authenticated cell lines avoid studying the wrong cells by mistake.

   • Cytion STR-authenticated human cell lines
   • Serana DMEM, high glucose, with L-glutamine and sodium pyruvate MCL-002-500ML
   • Serana Fetal+ FBS, triple 0.1 µm filtered, EU eligible S-FBSP-EU-015
6. 6

   Check that it worked

   Measure the gene's message (RNA by qPCR) and its product (protein by Western blot or flow cytometry), and compare 'on' with 'off'.

   • Takara Bio PrimeScript RT Reagent Kit with gDNA Eraser RR047A
   • Takara Bio TB Green Premix Ex Taq II (Tli RNase H Plus) RR820A
7. 7

   Keep cultures clean optional

   Mycoplasma are tiny bacteria that infect cell cultures invisibly and change how cells behave.

   • Takara Bio TaKaRa Mycoplasma qPCR Detection Kit RR277A

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CRISPR is a search-and-cut tool. The guide RNA is the address, Cas9 is the scissors. The cell repairs the cut quickly and sloppily, which switches the gene off (knock-out), or neatly copies a template you supply, which writes in a change (knock-in).

Typical customers: Molecular and cell biology labs, disease-modelling groups, stem-cell and gene-therapy researchers

1. 1

   Choose the target and design guides

   Free online tools suggest guide RNAs for your gene. Order two or three and test which one cuts best before committing to cells.

   • Takara Bio Guide-it sgRNA Screening Kit 632639
2. 2

   Make the guide and the scissors

   Either make guide RNA and add purified Cas9 protein (a 'ribonucleoprotein', RNP), or put both on one plasmid or virus.

   • Takara Bio Guide-it sgRNA In Vitro Transcription Kit 632635
   • Takara Bio Guide-it Recombinant Cas9 (Electroporation-Ready) 632641
3. 3

   Add a repair template (knock-in only) optional

   For a precise change or tag, supply DNA that carries the new sequence flanked by matching 'arms'. The cell copies it while repairing the cut.

   • Takara Bio Guide-it Long ssDNA Production System v2 632666
4. 4

   Deliver into cells

   Electroporation (a brief electric pulse) carries RNP into most cells; chemical transfection suits plasmids in cell lines; virus reaches primary cells.

   • Takara Bio Xfect Transfection Reagent 631317
5. 5

   Find the edited cells

   Simple gel-based kits show whether cutting happened in a pool of cells, then which single-cell clones carry the edit.

   • Takara Bio Guide-it Mutation Detection Kit 631443
6. 6

   Prove the edit by sequencing

   Amplify the edited region with a high-accuracy enzyme and read it.

   • Takara Bio PrimeSTAR Max DNA Polymerase R045A
   • Microsynth Sanger sequencing
7. 7

   Grow single-cell clones

   Each edited cell is grown into a colony so every cell in the final line carries the same edit.

   • Takara Bio Cellartis iPSC Single-Cell Cloning DEF-CS Culture Media Kit Y30021
8. 8

   Genome-wide screen optional

   Instead of one gene, a library knocks out every gene across thousands of cells to find which ones matter, for example for drug resistance.

   • Takara Bio Guide-it CRISPR Genome-Wide sgRNA Library System 632646

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Induced pluripotent stem cells (iPSCs) are like students who have not chosen a career yet: they can still become almost any cell. Good culture keeps them undecided and healthy; differentiation is the training course that turns them into liver, heart or other cells. Mesenchymal stem cells (MSCs) from bone marrow, fat or umbilical cord are a different, more limited kind used widely in regenerative medicine.

Typical customers: Stem-cell and regenerative-medicine centres, disease-modelling and toxicology labs, translational groups

1. 1

   Source the starting cells

   Start from established stem-cell lines, from primary MSCs, or from a patient's own cells.

   • Procell Human bone-marrow and umbilical-cord MSCs and primary cells
2. 2

   Keep stem cells healthy and undecided

   A defined culture system feeds the cells and lets you split them as single cells without them changing.

   • Takara Bio Cellartis DEF-CS 500 Culture System Y30010
3. 3

   Engineer the cells optional

   Add a reporter, knock out a disease gene or correct a mutation, usually before differentiation.

   • Takara Bio Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System 632643
4. 4

   Turn them into tissue cells

   A timed series of media pushes the stem cells step by step into the target cell type, then a maintenance medium keeps them working.

   • Takara Bio Cellartis Hepatocyte Differentiation Kit Y30050
5. 5

   Grow MSCs for regenerative work optional

   MSCs are expanded in their own medium, ideally without animal serum for any clinical direction.

   • Takara Bio Cellartis MSC Xeno-Free Culture Medium Y50200
6. 6

   Check identity and quality

   Confirm the cells are what you think: markers by flow cytometry or qPCR, a normal chromosome count, and no mycoplasma.

   • Elabscience Flow-cytometry antibodies (for example anti-human CD34, CD45) E-AB-F1324A
   • Takara Bio PrimeScript RT Reagent Kit with gDNA Eraser RR047A
   • Takara Bio TaKaRa Mycoplasma qPCR Detection Kit RR277A
7. 7

   Move towards the clinic (GMP) optional

   For therapies, reagents must be made under GMP with full documentation, and the product needs release testing.

   • Takara Bio Cellartis DEF-CS 500 Xeno-Free GMP Grade Basal Medium Y30071

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Take a patient's own immune cells, give them a new 'radar' gene so they recognise the cancer, grow them into an army, and check they are safe before they go back. Takara supplies the delivery and growth steps used in many clinical programmes; the other steps come from our cell-culture and QC partners.

Typical customers: Haematology and oncology research centres, cell-therapy and translational units, biotech

1. 1

   Collect and separate white cells

   Mononuclear cells are separated from blood by density-gradient centrifugation.

   • PAN-Biotech Pancoll human, density 1.077 g/ml P04-60100
2. 2

   Wake up (activate) the T cells

   T cells only take up a gene and multiply after they are activated.

   • Takara Bio Anti-CD3 mAb (GMP) T210
   • Takara Bio LymphoONE T-Cell Expansion Xeno-Free Medium WK552S
3. 3

   Build and produce the gene vector

   The CAR gene is cloned into a lentiviral vector, then producer cells make virus that is concentrated and counted (titre).

   • Takara Bio Lenti-X Packaging Single Shots (VSV-G) 631275
   • Takara Bio Lenti-X 293T Cell Line 632180
   • Takara Bio Lenti-X qRT-PCR Titration Kit 631235
4. 4

   Put the gene into the T cells (transduction)

   RetroNectin coats the plate and holds virus and cells together, so the gene goes in efficiently with less virus.

   • Takara Bio RetroNectin (recombinant human fibronectin fragment) T100A
5. 5

   Grow the cells

   The engineered cells are expanded for one to two weeks.

   • Takara Bio LymphoONE T-Cell Expansion Xeno-Free Medium WK552S
6. 6

   Test quality and potency

   Check how many copies of the gene went in, that the cells kill or respond to their target, and that the product is free of mycoplasma and residual DNA.

   • Takara Bio Lenti-X Provirus Quantitation Kit 631239
   • Takara Bio TaKaRa Mycoplasma qPCR Detection Kit RR277A
7. 7

   Freeze and store

   Cells are frozen in a protein-free medium and stored in liquid nitrogen until use.

   • PAN-Biotech Cryopan II, chemically defined, protein-free freezing medium P07-93010

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A viral vector is a delivery van: the virus's own genes are removed and replaced with yours, so it can carry the gene into cells but cannot multiply. Lentivirus suits dividing and non-dividing cells in a dish and integrates for long-term expression; AAV is the usual choice for delivery in animals and patients.

Typical customers: Gene-therapy, neuroscience and vaccine research groups, vector cores

1. 1

   Clone the gene into a transfer plasmid

   The gene goes into a plasmid with the signals that let it be packaged into the virus.

   • Takara Bio In-Fusion Snap Assembly Master Mix 638947
   • Takara Bio Stellar Competent Cells 636763
2. 2

   Make the virus

   Producer cells receive the transfer plasmid plus helper plasmids and release virus into the medium.

   • Takara Bio AAVpro Helper Free System (AAV2) 6230
   • Takara Bio AAVpro 293T Cell Line 632273
3. 3

   Purify and concentrate

   Virus is separated from cell debris and concentrated.

   • Takara Bio AAVpro Purification Kit Maxi (All Serotypes) 6666
4. 4

   Count the virus (titre)

   qPCR counts the genome copies so every experiment gets a known dose.

   • Takara Bio AAVpro Titration Kit (for Real Time PCR) Ver.2 6233
5. 5

   Quality control optional

   Check for mycoplasma and leftover producer-cell or plasmid DNA.

   • Shentek Bio SHENTEK Residual HEK293 DNA Quantitation Kit (3G) 1101104-1
   • Takara Bio TaKaRa Mycoplasma qPCR Detection Kit RR277A

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