Mutation Generation & Detection HSC Cores: Home

Lonzas 4D Nucleofector Technology is an improved electroporation technology that can help researchers achieve high transfection efficiencies in standard cell lines, primary cells, stem cells, and hard to transfect cell lines. With the 4D Nucleofector high efficiencies can be reached using much lower substrate amounts and with moderate impact on viability. The comprehensive way in which 4D Nucleofector Programs and cell type-specific solutions are developed enables nucleic acid and protein substrate delivery not only to the cytoplasm, but also through the nuclear membrane and into the nucleus. This allows for high efficiencies up to 99% and makes the transfection success independent from cell proliferation.

The MGD Core has acquired a complete Lonza 4D Nucleofector System that any and all researchers can use. This complete System is made up of four unique functional parts:

Core Unit The main control center for the 4D-Nucleofector System that controls the function of all other units. It has a 5.7 touch screen to operate all units and is loaded with intuitive operation software for designing and saving individual experimental setups.

X Unit This base unit allows Nucleofection of cells in suspension in 20ul Nucleocuvette 16-well strips or in single-use 100ul Nucleocuvettes. Each well in a 16-well Nucleocevette strip is electroporated independently allowing for different conditions to be tested and re-use of the strips if wells are not used. This unit is perfect for testing individual conditions on cells and for small-scale experiments.

Y Unit This unit allows Nucleofection of cells while still adherent to 24 well culture plates. This unit is perfect for working with adherent cells, such as neurons derived from stem cells, which are not transfectable in suspension. Transfection of adherent cells using the Y unit may lead to more physiological response in cells.

96-well Shuttle Controlled by the Core unit the 96-well Shuttle is an add-on unit that allows for convenient optimization of conditions or large-scale screens to be preformed. Each individual well is processed independently allowing 96 different experimental conditions to be tested at one time.

The full Lonza 4D Nucleofector System is housed in Room 7470 of the Eccles Institute of Human Genetics, along with a cell culture hood for researchers to work with their cells in and a 37C incubator to store their cell.

To reserve time to use the Lonza 4D Nucleofector System please use the following link to login to the HSC Core Research Facilities resource page. Select the Mutation Generation page option and then select the 4D Nucleofector page option. Here you can reserve time to use the System. Researchers will be charged a $5.00 fee for every 30 minutes block reserved. Individual experiments do not require more than one 30-minute block.

University of Utah Core Labs

Lonza maintains two databases with protocols (including Nucleofector Solution types and program numbers) of optimized protocols for a wide range of cell lines. Basically Lonza has already done the optimization experiment and determined the best conditions to achieve the highest transfection efficiency with the least amount of cell death. These databases are a good starting place to determine the most optimal protocol for working with your specific cell line.

Lonzas public optimized cell line protocol database can be found at the following link: http://bio.lonza.com/6.html

Lonza also maintains a database of user-optimized protocols that is not publicly available. Please contact either Dr. Gregory Alberts or Haylee Erickson at the following information for access. Dr. Alberts is an expert on the use of the 4D Nucleofector System and a great resource for the best transfection protocols to use with your specific cell line. The following is a link to a seminar given by Dr. Alberts on using the 4D Nucleofection system:Dr. Alberts 4D Nucleofection System

Gregory Alberts, Ph.D. gregory.alberts@lonza.com

Global Subject Matter Expert

Lonza Pharma Bioscience Solutions

Haylee Erickson haylee.erickson@lonza.com

Sales Specialist, Rocky Mnt/Pacific NW

Lonza Pharma Bioscience Solutions

Original CRISPR-Cas9 experiments were performed using DNA vectors, viral vectors or RNA transfection to produce the components of the system: Cas9 protein and single guide RNAs (sgRNAs). New advances have demonstrated that these component can be produced and combined in vitro to form a ribonucleoprotein complex or RNP that is functional in vitro and in vivo without the need for transcription or translation. This CRISPR RNP complex a can be delivered directly to cells and results in immediate, efficient, and specific target cleavage by the CRISPR RNP.

Several labs have shown that combining the CRISPR RNP approach with the extremely high transfection efficiency of the Lonza 4D Nucleofection System can result in mutation frequencies reaching 90% of targeted gene copies in several different cell types. CRISPR RNP delivery is applicable to a wide range of cell types, including established cell lines, primary cells, adherent cells such as primary neurons, iPCS, and stem cells. With these cell types using the CRISPR RNP approach can dramatically shortened the time it takes to create targeted variants of your gene of.

Please contact the MGD Core if you have any questions concerning this approach or would like to discuss the possibility of using CRISPR RNP in your research. Also, the following link is a generalized protocol detailing how to combine the CRISPR RNP approach with the Lonza 4D Nucleofection System.

General Nucleofection Protocol

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Mutation Generation & Detection HSC Cores: Home