Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: “transformation” is typically used to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells, including plant cells.- Wikipedia

Transfection, technique used to insert foreign nucleic acid ( DNA or RNA) into a cell, typically with the intention of altering the properties of the cell. The introduction of nucleic acid from a different cell type can be accomplished using various biological, chemical, or physical methods. - Britannica Transfection is the process of introducing foreign DNA into the cells either by physical (electroporation) or chemical (cationic lipid or calcium phosphate reagents) methods. From: Stem Cell Manufacturing, 2016

Transfection is a modern and powerful method used to insert foreign nucleic acids into eukaryotic cells. The ability to modify host cells' genetic content enables the broad application of this process in studying normal cellular processes, disease molecular mechanism and gene therapeutic effect. National Institutes of Health Apr 21, 2021 ¹⁾

Cornel Mülhardt, E.W. Beese M.D., in Molecular Biology and Genomics, 2007 Transfection through Bombardment: Particle Delivery

Transfection through bombardment was originally developed for the transfection of plant cells, but it is now also used for mammalian cells. Small wolfram or gold particles are coated with DNA and then shot onto the cells at a high speed.

This can be performed on cells in culture and in the living animal, such as cells of the liver, skin, and spleen. The depth of penetration is slight so that the transfection results only on the surface. The instrument required for this method can be obtained from BioRad under the martialistic name of Helios Gene Gun System.

Advantage: This method functions quite well where other methods fail, because the DNA is “forced” into the cells.

Disadvantage: Extensive manipulation, large preparations, and lots of time are needed. The efficiency of the transfection is frequently not particularly high.²⁾

Pages 169-221 Publisher Summary

During investigation, the function of deoxyribonucleic acid (DNA) sequences must indirectly demonstrate expression by means of the existence of messenger ribonucleic acid (mRNA) by ribonuclease protection, in situ hybridization, or in situ polymerase chain reaction (PCR).

The next step consists of cloning the DNA in an expression vector—a plasmid that, in addition to the usual plasmid components such as the bacterial replication origin and antibiotic resistance, contains a gene with a promoter or a coding area.

Depending on whether the experimenters want to examine a promoter or a complementary DNA (cDNA), they replace the region of the DNA that interests them. A vector is chosen with an easily demonstrable reporter gene, before which the experimenter can insert the promoter. Enhancer sequences can also be explored in this manner by using a vector that contains a basic promoter and whose activity in the cloning of regulatory elements can be upregulated or downregulated.

When determining the section in which the DNA activity can be found, mutagenesis can be used to remove sequences. The choice of a proper reporter gene is decisive in this kind of investigation. When working with cDNA, experimenters have a multitude of expression vectors available to them that deliver a promoter and consequently wait to be associated with an open-reading pattern.

Viral promoters such as that of the cytomegalovirus or from simian virus 40 permit a quite intense expression in mammalian cells, and functional investigations can be frequently accomplished immediately. Cell-typical promoters permit expression based on certain tissue limitations, such as in the manufacture of transgenic animals, and controllable promoters already exist.

Copyright © 2007 Elsevier Inc. All rights reserved.³⁾

Author links open overlay panelX.BaoS.P.Palecek

Chapter 1 - Genetic Engineering in Stem Cell Biomanufacturing

Abstract

Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells (hiPSCs) offer unprecedented opportunities to study human organogenesis, model human disease, and provide unlimited cell sources for regenerative medicine.

To realize their full potential, however, genetic manipulation strategies with high specificity and efficiency are required. Genetic manipulation is an important tool to optimize conditions for direct differentiation of hPSCs toward specific lineages, and to correct genetic mutations for clinical application of patient-specific hiPSCs.

This chapter briefly summarizes a variety of genetic manipulation strategies used to generate useful hPSC lines, and reviews their advantages and shortcomings to provide insights on suitable approaches for stem cell biomanufacturing applications.⁴⁾

CRISPR/Cas9 - Genome editing - Human pluripotent stem cells - TALEN - Zinc-finger nuclease

Abstract Graphene as a star in materials research has been attracting tremendous attentions in the past few years in various fields including biomedicine. In this work, for the first time we successfully use graphene as a non-toxic nano-vehicle for efficient gene transfection.

Graphene oxide (GO) is bound with cationic polymers, polyethyleneimine (PEI) with two different molecular weights at 1.2 kDa and 10 kDa, forming GO-PEI-1.2k and GO-PEG-10k complexes, respectively, both of which are stable in physiological solutions.

Cellular toxicity tests reveal that our GO-PEI-10k complex exhibits significantly reduced toxicity to the treated cells compared to the bare PEI-10k polymer. The positively charged GO-PEI complexes are able to further bind with plasmid DNA (pDNA) for intracellular transfection of the enhanced green fluorescence protein (EGFP) gene in HeLa cells.

While EGFP transfection with PEI-1.2k appears to be ineffective, high EGFP expression is observed using the corresponding GO-PEI-1.2k as the transfection agent. On the other hand, GO-PEI-10k shows similar EGFP transfection efficiency but lower toxicity compared with PEI-10k.

Our results suggest graphene to be a novel gene delivery nano-vector with low cytotoxicity and high transfection efficiency, promising for future applications in non-viral based gene therapy. ⁵⁾

Substack December 16,2022 by Jessica Rose Please refer to the published work in the journal ‘Nanoscale’ entitled: “Functionalized graphene oxide serves as a novel vaccine nano-adjuvant for robust stimulation of cellular immunity”.

This work was published in 2016 by a group in China and let me tell you, they did a lot of work and asked a lot of questions. Their methods are so good, that I could actually reproduce their work.

Their primary goal was to find out if their adjuvant antigen delivery system, graphene oxide-polyethylene glycol-polyethylenimine (GO-PEG-PEI), would be functional with regard to specific immune stimulation, and/or less toxic than aluminum-based adjuvants.

Their results showed that a PEG-PEI coated graphene oxide sheet - which incidentally really loves absorbing antigens like Ure B (an antigen associated with a bacterium called Helicobacter pylori) - was really good at trafficking antigen to dendritic cells to induce their maturation and activation when this concoction was injected intradermally.

What I really need to get my hands on are the original methods… this isn’t even locatable using sci-hub. Anyone crafty enough to get this paper? I have the supplementary materials but not the text and will not be providing personal details to ‘sign up’ for anything to read science. It’s against my code

Summary of paper translated into english: PEG-PEI-conjugated GO sheets used as adjuvants to deliver immune-stimulating agents like bacterial proteins, are a thing. ⁶⁾