📖 Lectura:

*Gene transfer allows researchers to manipulate and study specific genes (and their protein products), or noncoding regions in isolation. A plasmid, or vector, is a small circular DNA molecule that replicates independently of the chromosomal DNA. In gene transfer, scientists can use the plasmid molecules to provide a "folder" in which to insert a desired DNA fragment. Plasmids are usually introduced into a bacterial host for proliferation. In the bacterial context, scientists call the DNA fragment from the human genome (or the genome of another studied organism) foreign DNA, or a transgene, to differentiate it from the bacterium's DNA, or the host DNA.

Plasmids occur naturally in bacterial populations (such as Escherichia coli) and have genes that can contribute favorable traits to the organism, such as antibiotic resistance (the ability to be unaffected by antibiotics). Scientists have repurposed and engineered plasmids as vectors for molecular cloning and the large-scale production of important reagents, such as insulin and human growth hormone. An important feature of plasmid vectors is the ease with which scientists can introduce a foreign DNA fragment via the multiple cloning site (MCS). The MCS is a short DNA sequence containing multiple sites that different commonly available restriction endonucleases can cut. Restriction endonucleases recognize specific DNA sequences and cut them in a predictable manner. They are naturally produced by bacteria as a defense mechanism against foreign DNA. Many restriction endonucleases make staggered cuts in the two DNA strands, such that the cut ends have a 2- or 4-base single-stranded overhang. Because these overhangs are capable of annealing with complementary overhangs, we call them “sticky ends.” Adding the enzyme DNA ligase permanently joins the DNA fragments via phosphodiester bonds. In this way, scientists can splice any DNA fragment generated by restriction endonuclease cleavage between the plasmid DNA's two ends that has been cut with the same restriction endonuclease.

Plasmids with foreign DNA inserted into them are called recombinant DNA molecules because they are created artificially and do not occur in nature. They are also called chimeric molecules because the origin of different molecule parts of the molecules can be traced back to different species of biological organisms or even to chemical synthesis. We call proteins that are expressed from recombinant DNA molecules recombinant proteins. Not all recombinant plasmids are capable of expressing genes. The recombinant DNA may need to move into a different vector (or host) that is better designed for gene expression. Scientists may also engineer plasmids to express proteins only when certain environmental factors stimulate them, so they can control the recombinant proteins' expression.*

📽️ Video del proceso

https://content.dnalc.org/content/c15/15476/recombination.mp4

Esta imagen resume el proceso general a detalle, usando como ejemplo la inserción de resistencia a antibiótico:

📖 En Resumen:

• Genetic modification, or genetic engineering, involves gene transfer between species.
• Genes may be transferred to bacterial plasmids, restriction endonucleases and DNA ligase.
• The bacterial plasmids are then used as vectors to deliver new genes into organisms. Other vectors might be viruses or a recombinant cell.
• Restriction endonucleases are enzymes that digest DNA strands at specific sequences, leaving behind single-stranded ‘sticky ends’.
• Restriction endonucleases are enzymes that digest DNA strands at specific sequences, leaving behind single-stranded ‘sticky ends’.
• The desired gene is mixed with bacterial plasmids that have been cut with endonucleases. The sticky ends bind by complementary base pairing to the inserted gene. DNA ligase enzyme joins the DNA backbones to produce a recombinant plasmid.
• The recombinant is cloned in small populations and then on a larger scale to produce the desired gene product.