셀타젠 Genetic Genotyping

This week we will talk about the transformation of human fibroblasts into hepatocytes using the biotechnological techniques: infection with lentiviral vectors and cellular immortalization. The purpose? Study a situation with a high idiosyncratic component: the damage that causes the liver of each person and the consumption of medicines. The authors? The Experimental Hepatología Unit of the Instituto de Investigación Sanitaria La Fe de Valencia… FOLLOW LEYENDO!

What does this strategy consist of?

From somatic cells (any cell in our body except sexual cells) of the individual in the studio, and transform them into cells with a hepatocyte-like phenotype (which in English is called hepatocyte-like cells) to be able to work with them in vitro form.

For him, we have to speak of the strategies: cellular immortalization and lentiviral infection; and some concept like: cell line or primary cultivation.

If we put ourselves in context, to study the liver damage of a patient the most obvious would be to show hepatocytes of the patient and cultivate them. Using cells obtained directly from an organ, the fabric is called primary culture. What is your main disadvantage? That in vitro growth is very limited. This is how it is difficult to extract hepatocytes from a patient.

Not only will you finish imagining it, the habitants will sometimes work with cell cultures. When we were able to grow cells, we were able to extend them on different “platitos” and wait for them to grow to try them out and keep them in cultivation, turning them on another plate or plate. In vitro, it is called in vitro by the original material of these plates, the glass.

How can it be an alternative to these primary crops?

Realize a transformation or cellular immortalization. To decide, to co-operate this primary culture and to induce some change so that these cells can multiply indefinitely, let us turn them to death. In this case, we would be talking about a cell line. A set of cells that you can maintain indefinitely in cultivation to work with them comfortably.

Do you disadvantages? The immortalization process could change its phenotype (the observable characteristics derived from combining genes and the environment), and they are not identical to the original cells.

How can a cell be immortalized? It may seem like a twist, but there are only so many strategies. In this case we will focus on the method followed by these researchers: using the SV40 T large antigen.

SV40 is a virus capable of infecting both humans and humans. What is interesting? A gen that houses its genome. This gene produces the protein known as the large SV40 antigen. This protein is able to block the p53 action.

We have also spoken on other occasions of this tumor suppressor, we have a small reminder. p53, known as the guardian of the genome, is the protein responsible for detecting irreparable damage in the genome of other cells and activating its mutation.

In this way, it avoids that damage in the DNA (which could end up leading to mutations and tumors, simply due to the aging of the cell) is not transmitted to new cells.

Who can get the large antigen from the SV40? One joins an essential region for the function of p53, completely blocking its action. So, this cell will be unable to detect the changes in the genome and it will never die, becoming literally and immortal.

Of course, p53 is known as a tumor suppressor because it stops the accumulation of mutations that could lead to cancer. Unfortunately, there are only one of the main genes that appear to be altered in tumor cells, which we think are also deadly.

We will now go to lentiviral infection. This concept is easily understandable if we understand how a virus works. A virus comes into contact with a cell and inserts its genetic material.

In this way, the DNA (the RNA) of the virus multiplies using our own cells, so that it can continue to infect. Well, when we refer to lentiviral infection, we are talking about cogerating these viruses, making changes in their genome so that in the future this ability to jump from cell to cell, and to change our genes or genes of interest.

How did we get there? That using a virus-friendly mechanism, we introduce into the genome of cells the genes that interest us.

And the concepts are cleared, now we can understand the work of this group of researchers: Once the patients’ fibroblasts were isolated, these cells were subjected to lentiviral infections.

Firstly, to introduce the SV40 large T antigen into its genome and immortal cells to easily work in vitro. And secondly, with a cocktail of genes needed to express a hepatocyte phenotype.

What gene cocktail? Genes for three transcription factors: HNF4A, HNF1A and FOXA3. The factors of transcription in proteins are charges to induce the expression (transformation into protein) of other genes.

These 3 transcription factors are key to expressing essential genes for a liver phenotype. In short, these transcription factors induce the expression of genes that provide a cell with the proper characteristics of a liver cell.

More details? These researchers used an inducible transcription factor expression system. Decide, these factors will only be expressed, and will transform the cell into hepatocyte, when we want it. As? Using a doxycycline-activated promoter.

Very straightforward: the sequence of any genre preceded by another sequence, the promoter. This promoter is responsible for allowing, however, the expression of the genes that they have by default. It is precisely in this region from which the transcriptional factors are united to activate the expression of the genes they control.

In this specific system, the 3 transcription factors have been inserted into the fibroblast genome with a forward promoter. Promoter that only activates and induces the expression of two factors when researchers add to the doxycycline cultivation.

If this antibiotic is not present, the cells follow the fibroblast and can be maintained indefinitely in culture. If doxycycline is used, the transcription factors are expressed and can, in turn, induce the expression of the genes that “transform” the fibroblast into hepatocyte.

The result? Cells owned by a patient (with their genome), easily isolated (from the skin), immortalized (never dying) and with characteristics of the same patient’s hepatocytes (as a result of these three genes).

Thanks to this strategy, it is possible to work in vitro with the individual cells of each patient, avoiding the use of primary cultures and their disadvantages, and I could study the characteristic changes of each patient in response to the liver by drugs.

With the arrival of winter, concerns about the transmission of several viruses, especially the flu, increase. When people think of viruses, they quickly associate them with diseases or chemical weapons. However, for us, biotechnologists, they can also be important tools, as we can use their ability to infiltrate and exploit living systems in favor of society. Calm down, I’ll explain to you how this is possible!

Well, initially it is necessary for you to understand that viruses are basically composed of a genetic material inside a protein capsule, thus, they are not considered living beings, as they cannot replicate by themselves. Once they infect cells that allow them to replicate, they lose their identity of origin and act under viral command.

Now that you know how viruses replicate, it’s important to know that even bacteria are targets for these smarties: phages (or bacteriophages) are viruses that only infect bacteria. This is exactly where biotechnology comes in, and we think: Now, if phages are capable of killing bacteria, it can become an advantage for humans, who can use them to kill unwanted bacteria.

Since then, they have been extensively researched for complementary use in antibiotic therapy, since they are immune to the resistance mechanism and specific to their host (each phage only infects one strain of bacteria). This feature prevents the “beneficial” bacteria from being destroyed. However, in order to use this treatment it is necessary to carry out laboratory tests to identify precisely which species of bacteria caused the infection. Studies are also needed to monitor, in the long run, whether bacteria can evolve into phage resistance. However, when bacteria become resistant to one phage, they become susceptible to another. In this way there is almost an endless supply of possible new treatments.

Tumor-targeted viruses
Until recently, there was no way to kill cancer cells without harming healthy cells in the rest of the body, due to the difficulty of targeting a treatment that affected only those affected. However, biotechnology has revolutionized this scenario by developing viruses that selectively destroy tumors. Amgen’s T-VEC is a herpes oncolytic virus, genetically modified to treat melanoma, killing cancer cells in the skin without affecting healthy cells. This is possible because the modified herpes virus can only replicate within cancer cells. They are injected directly into the tumor until the cancer subsides, for approximately four months.

In Germany, ParvOryxo was developed, which selectively kills tumor cells from a wide variety of tumors including glioblastoma and pancreatic cancer. It is able to pass the blood-brain barrier, which protects the brain, directly killing tumor cells, in addition to altering the tumor’s microenvironment, making it more visible to the immune system and increasing its vulnerability to immuno-oncology approaches.

“Pigeons carrier” of genetic material (viral vectors)
 When removing the pathogenic components of the virus genome, they can be used as true “pigeons”, delivering genes of interest in genetic therapies. Luxturna, for example, is a viral vector that carries a functional copy of the RPE65 gene in retinal cells, restoring the vision of patients with progressive vision loss due to a mutation.

Although its sinister reputation can never be completely erased, there is no denying that viruses are far from just carrying disease and death. They can act as fantastic biotechnological tools, capable of providing powerful treatments that would be impossible without your help.

There are many methods consist on the identifcation of the repiratory virus.

Viral respiratory infection can be caused by nemerous viruses:

  • RSV A and RSV B (Respiratory Syncytial Virus).
  • Parainfluenza Virus (types 1–4).
  • Numerous Adenoviruses.
  • Rhinoviruses.
  • metapneumovirus.
  • Bocavirus.
  • Avian Influenza Virus (H5N1 and H7N9).
  • Coronaviruses; including SARS (Severe Acute Respiratory Virus) and SARS-COV2 (The novel coronavirus which is responsible for COVID-19.
SARS- CoV 2 Structure. Contributed by Rohan Bir Singh, MD; Made with Biorender.com

Respiratory Infection

The above listed viruses causes different respiratory infection with symptoms from mild to severe, and thus form runny nose and sneezing to pharyngitis, laryngitis, bronchitis, or pneumonia.

The severity of disease varies depending on the level of immunity of the individual.

Diagnosis methods

There are many methods for the diagnosis of the respiratory viruses; we can say that there is traditional methods and molecular methods.

The traditional methods are:

Cell Culture

This method consist on:

  • Isolation of the virus in cell cultures
  • Incubation of specimen with these cell cultures in tubes.
  • Placing the tubes in roller drum (Rotate for almost 10 days).
  • Observing daily the cells under the microscopes, the damaged cells indicate the presence of the virus.

The inconvenient of this method is that not all viruses are culturable, and this method is not sensitive enough when antibodies in the specimen neutralize the virus.

Direct Fluorescent Antibody (FDA)

This methode consit on staining or the presence of virus-infected cells.

  • collection of epithelial cells from a nasopharyngeal swab.
  • fixing the epithelial cells to a glass microscope slide.
  • Staining with individual antibodies labeled with a fluorescent tag.
  • Viewing the slide with a fluorescent microscope.
Left: positive; showis the presence of virus-infected cells (green) stained with a fluorescent monoclonal antibody.
Right: negative shows uninfected cells stained with a red counterstain.

Shell Vial Culture

This method consist on:

  • Inoculation of an aliquot of the specimen onto a preformed cell monolayer in a small vial containing a mixture of two susceptible cells.
  • Centrifugation to enhance virus attachment and entry.
  • The centrifugation-assisted inoculation of the cells increases the amount of viral proteins produced.
  • This allows staining to be performed at 24–48 h and thus providing a test result to be obtained significantly earlier than the 7–10 days necessary for traditional cell culture.

Enzyme-linked immunosorbent assays (ELISA)

The rapid enzyme-linked immunosorbent assays (ELISAs) consist on using a monoclonal conjugated antibody to an enzyme to quantify and detect the presence of a specific antigen in a sample.

The molecular methods are:

Nucleic Acid Amplification Tests (NAAT)

This technique started in the late 1980s, developped for the the first time for the influenza, In 1983; 1983 by Kary B. Mullis ( used a nucleic acid amplification method called Polymerase Chain Reaction (PCR).

Within a decade, NAATs were developed for all of the respiratory viruses.

The most used technique is PCR, but there are other techniques:

  • Strand Displacement Amplification (SDA).
  • Nucleic Acid–Sequence-Based Amplification (NASBA).
  • Transcription-Mediated Amplification (TMA).
  • Loop-Mediated Isothermal Amplification (LAMP).

These all NAATs techniques consist on:

  • Extraction of the nucleic acid from the respiratory tract specimen.
  • Copying the viral ribobucleic acid (RNA) into a complementary deoxyribonucleic acid (cDNA) by the enzyme Reverse transcriptase.
  • Amplifying the cDNA by PCR using a virus-specific oligonucleotide primers.
  • The result is a billion copies of DNA.
  • These DNAs can then easily detected by different laboratory methods.

Molecular methods are more sensitive than traditional methods, they are also efficient and give results in a very short time.