Information

7.14A: Putting Foreign DNA into Cells - Biology


The methods used to get DNA into cells are varied (e.g., transformation, transduction, transfection, and electroporation).

LEARNING OBJECTIVES

Describe the methods of introducing foreign DNA into cells

Key Points

  • When microorganisms are able to take up and replicate DNA from their local environment, the process is termed transformation.
  • In mammalian cell culture, the analogous process of introducing DNA into cells is commonly termed transfection.
  • Electroporation uses high voltage electrical pulses to translocate DNA across the cell membrane (and cell wall, if present).

Key Terms

  • transformation: The alteration of a bacterial cell caused by the transfer of DNA from another, especially if pathogenic.
  • microorganisms: A microorganism or microbe is a microscopic organism that comprises either a single cell (unicellular), cell clusters, or multicellular relatively complex organisms.

The DNA mixture, previously manipulated in vitro, is moved back into a living cell, referred to as the host organism. The methods used to get DNA into cells are varied, and the name applied to this step in the molecular cloning process will often depend upon the experimental method that is chosen (e.g., transformation, transduction, transfection, electroporation).

When microorganisms are able to take up and replicate DNA from their local environment, the process is termed transformation, and cells that are in a physiological state such that they can take up DNA, are said to be competent. In mammalian cell culture, the analogous process of introducing DNA into cells is commonly termed transfection. Both transformation and transfection usually require preparation of the cells through a special growth regime and chemical treatment process that will vary with the specific species and cell types that are used.

Electroporation uses high voltage electrical pulses to translocate DNA across the cell membrane (and cell wall, if present). In contrast, transduction involves the packaging of DNA into virus-derived particles, and using these virus-like particles to introduce the encapsulated DNA into the cell through a process resembling viral infection. Although electroporation and transduction are highly specialized methods, they may be the most efficient methods to move DNA into cells.

Whichever method is used, the introduction of recombinant DNA into the chosen host organism is usually a low efficiency process; that is, only a small fraction of the cells will actually take up DNA. Experimental scientists deal with this issue through a step of artificial genetic selection, in which cells that have not taken up DNA are selectively killed, and only those cells that can actively replicate DNA containing the selectable marker gene encoded by the vector are able to survive.

When bacterial cells are used as host organisms, the selectable marker is usually a gene that confers resistance to an antibiotic that would otherwise kill the cells, typically ampicillin. Cells harboring the vector will survive when exposed to the antibiotic, while those that have failed to take up vector sequences will die. When mammalian cells (e.g., human or mouse cells) are used, a similar strategy is used, except that the marker gene (in this case typically encoded as part of the kanMX cassette) confers resistance to the antibiotic Geneticin.


Question : A transformation is: a. Fusing two bacterial cells together b. Introducing plasmid DNA into mammalian cells c. Introducing foreign DNA into a bacterial cell d. Introducing a bacterial cell into a mammalian cell 2 After creating a recombinant vector, we will __________________ in this week's lab. a. Work with it without

After creating a recombinant vector, we will __________________ in this week's lab.

Work with it without any transformation

Transform it into mammalian cells

Transform it into yeast cells

Transform it into bacterial cells

3. The purpose of a transformation is:

Introduce new restriction sites into bacterial DNA

To create many bacterial cells

To create many copies of a recombinant vector

4. The most important factors for detecting which of the transformed bacteria have taken up the recombinant vector containing your gene of interest, are:

Use multiple cloning site

5. During transformation, heat shock is performed to:

To degrade the plasmid DNA

To degrade the bacterial DNA

To weaken the bacterial cell wall and make it permeable

6. After the incubation of competent cells, we put them under heat shock at what temperature?


Viral Transduction

Transduction is the insertion of foreign DNA into a cell via a virus (See Reference 1 and 2). Viruses are made of a protein coat that houses DNA within. Viruses can bind to living cells and inject their DNA. Or, viruses can push into the host as a membrane-bound vesicle, before releasing their DNA inside the host. The use of recombinant DNA technology can insert foreign DNA into host cells that are then purposefully infected with viruses. When the virus produces more of itself in the host cell, it also packages copies of the foreign DNA into the new viruses. When these new viruses burst out of the host cell, they are now carriers of the foreign DNA and can be used to introduce this DNA into other host cells.


The Process of Bacterial Transduction

Viruses cannot reproduce on their own. Instead, they must use the more advanced reproductive cell biology of the bacteria to make copies of themselves. To do that, bacteriophages hijack host cells.

When a bacteriophage encounters a bacterial cell, it binds to the cell and injects phage DNA through the plasma membrane into the cell. There, it takes command of the cell’s reproductive behavior. Instead of replicating its own genetic material, bacterium begins replicating new phage particles – components of virus cells.

The bacterial genes are degraded by the phages during this process. What is left of the bacterium is a replication machine for the virus.

The virus uses the bacterial cell to synthesize the protein scaffolding it needs for its components. Sometimes, it accidentally packages stray bacterial DNA into some of the phages along with the replicated viral DNA.

Once everything is ready, the virus lyses the bacterial cell. The bacterial cell bursts open, releasing the phages to bind to and infect other bacterial cells. Once bound, some of the phages will inject the bacterial genetic material they are carrying instead of viral DNA into the new bacterium.

Because the some of the phages are only carrying pieces of bacterial DNA, they cannot infect or lyse the new recipient cell. If the donor bacterial DNA fits into the new bacterial chromosome, the cell will express the genes as if they had always been there.


Sample 4 Lab 6a Transformation

Introduction:
Genes are transferred between bacteria by way of conjugation, transduction, or transformation. Conjugation takes place when the genetic material is transferred from one bacterium to another of a different mating type. Transduction requires the presence of a virus to act as a vector, or a carrier to transfer small pieces of the DNA from one bacterium to another. Transformation involves the transfer of genetic information into a cell by directly taking up the DNA. This lab uses transformation to insert a specific gene into a plasmid so that the cell takes on those characteristics for which the gene codes.

Plasmids are small rings of DNA that do carry genetic information. They can transfer genes, like genes for antibiotic resistance, which can occur naturally within them, or plasmids can act as carriers or vectors for introducing foreign DNA from other bacteria, plasmids, or even eukaryotes into recipient bacterial cells. Restriction endonucleases can be used to cut and insert pieces of foreign DNA into the plasmid vectors. If these plasmid vectors also carry genes for antibiotic resistance, transformed cells containing plasmids that carry the foreign DNA of interest in addition to the antibiotic resistance gene can be easily selected from other cells that do not carry the gene for antibiotic resistance. They are usually extrachromosomal. This means they exist separately from the chromosome. Some plasmids replicate only when the bacterial chromosome replicates, and usually exist only as single copies within the bacterial cell, but still others replicate on their own, autonomously. There can be anywhere from ten to two hundred copies within a single bacterial cell. There are specific plasmids called R plasmids that carry genes for resistance to antibiotics such as ampicillin, kanamycin, or tetracycline.

The bacterium Escherichia coli, or E. coli, is an ideal organism for the molecular geneticists to manipulate and has been used extensively in recombinant DNA research. It is a common inhabitant of the human colon and can easily be grown in suspension culture in a nutrient medium such as Luria broth, or in a petri dish of Luria broth mixed with agar, or nutrient agar. The single circular chromosome of E. coli contains about five million DNA base pairs, only one-six hundredth of the haploid amount of DNA in a human cell. Also, the E. coli cell may contain small plasmids, discussed earlier. The plasmids are broken up with calcium chloride, and the wanted gene is inserted and the bacteria can be grown on the nutrient or with an antibiotic to see if the gene has transformed the bacteria so that they are resistant to the antibiotics.

Materials:
The materials needed in this lab were two Luria agar plates, two Luria agar plates with ampicillin, two 15mL tubes, one inoculating loop, one bacterial spreader, several sterile micropipettes, calcium chloride, Luria broth, pAMP solution, a Bunsen burner, hotplate, ice, and a water bath.

Methods:
Mark one of the sterile 15mL tubes “+” and the other “-“, the plus tube obviously having the plasmid added to it while the other tube does not receive any. Using a sterile micropipette, add 250 microliters of ice cold 0.05M CaCl2 to each tube. Transfer a large 3mm colony of E. coli from the starter plate to each of the tubes using a sterile inoculating loop. Try to get the same amount of bacteria into each tube. Be careful not to transfer any agar. Vigorously tap the loop against the wall of the tube to dislodge the cell mass. Mix the suspension by repeatedly drawing in and emptying a sterile micropipette with the suspension. Add ten microliters of pAMP solution directly into the cell suspension in the tube labeled with a plus sign. Mix by tapping the tube. This solution contains the antibiotic resistance plasmid. Keep both of these tubes in ice for about 15 minutes. While the tubes are on ice, obtain two LB agar plates and two LB/Amp agar plates. Label each plate on the bottom as follows: one LB agar plate “LB+” and the other “LB-.” Label one LB/Amp plate “LB/Amp+” and the other plate “LB-.” A brief pulse of heat facilitates entry of foreign DNA into the E. coli cells. Heat-shock cells in both the + and – tubes by holding in a water bath of 42 degrees Celsius for ninety seconds. It is essential that cells be given a sharp and distinct shock so take the tubes directly from the ice to the water bath. Immediately return the tubes to the ice after ninety seconds. Use a sterile micropipette to add 250 microliters of Luria broth to each tube. Mix by tapping the tube. Any transformed cells are now resistant to ampicillin because they contain the gene. Place 100 microliters of the + cells on the LB+ plate and on the LB- plate, the other cells should be placed. Immediately spread the cells using a sterile spreading rod. This can be accomplished by running the rod through the Bunsen burner and allowing to cool by touching it to the agar on the part of the dish away from the bacteria. Spread the cells and once again run the rod through the fire to sterilize the rod. Allow the plates to set for several minutes, then tape the plates together and incubate inverted overnight.


Contents

Electroporation is performed with electroporators, purpose-built appliances that create an electrostatic field in a cell solution. The cell suspension is pipetted into a glass or plastic cuvette which has two aluminium electrodes on its sides. For bacterial electroporation, typically a suspension of around 50 microliters is used. Prior to electroporation, this suspension of bacteria is mixed with the plasmid to be transformed. The mixture is pipetted into the cuvette, the voltage and capacitance are set, and the cuvette is inserted into the electroporator. The process requires direct contact between the electrodes and the suspension. Immediately after electroporation, one milliliter of liquid medium is added to the bacteria (in the cuvette or in an Eppendorf tube), and the tube is incubated at the bacteria's optimal temperature for an hour or more to allow recovery of the cells and expression of the plasmid, followed by bacterial culture on agar plates.

The success of the electroporation depends greatly on the purity of the plasmid solution, especially on its salt content. Solutions with high salt concentrations might cause an electrical discharge (known as arcing), which often reduces the viability of the bacteria. For a further detailed investigation of the process, more attention should be paid to the output impedance of the porator device and the input impedance of the cells suspension (e.g. salt content).

Since the cell membrane is not able to pass current (except in ion channels), it acts as an electrical capacitor. Subjecting membranes to a high-voltage electric field results in their temporary breakdown, resulting in pores that are large enough to allow macromolecules (such as DNA) to enter or leave the cell. [13]

Additionally, electroporation can be used to increase permeability of cells during in Utero injections and surgeries. Particularly, the electroporation allows for a more efficient transfection of DNA, RNA, shRNA, and all nucleic acids into the cells of mice and rats. The success of in vivo electroporation depends greatly on voltage, repetition, pulses, and duration. Developing central nervous systems are most effective for in vivo electroporation due to the visibility of ventricles for injections of nucleic acids, as well as the increased permeability of dividing cells. Electroporation of injected in utero embryos is performed through the uterus wall, often with forceps-type electrodes to limit damage to the embryo. [14]

In vivo gene electrotransfer was first described in 1991 [15] and today there are many preclinical studies of gene electrotransfer. The method is used to deliver large variety of therapeutic genes for potential treatment of several diseases, such as: disorders in immune system, tumors, metabolic disorders, monogenetic diseases, cardiovascular diseases, analgesia…. [16] [17] [18]

With regards to irreversible electroporation, the first successful treatment of malignant cutaneous tumors implanted in mice was completed in 2007 by a group of scientists who achieved complete tumor ablation in 12 out of 13 mice. They accomplished this by sending 80 pulses of 100 microseconds at 0.3 Hz with an electrical field magnitude of 2500 V/cm to treat the cutaneous tumors. [19] Currently, a number of companies, including AngioDynamics, Inc. and VoltMed, Inc., are continuing to develop and deploy irreversible electroporation-based technologies within clinical environments.

The first group to look at electroporation for medical applications was led by Lluis M Mir at the Institute Gustave Roussy. In this case, they looked at the use of reversible electroporation in conjunction with impermeable macromolecules. The first research looking at how nanosecond pulses might be used on human cells was conducted by researchers at Eastern Virginia Medical School and Old Dominion University, and published in 2003. [20]

The first medical application of electroporation was used for introducing poorly permeant anticancer drugs into tumor nodules. [21] Soon also gene electrotransfer became of special interest because of its low cost, easiness of realization and safety. Namely, viral vectors can have serious limitations in terms of immunogenicity and pathogenicity when used for DNA transfer. [22]

A higher voltage of electroporation was found in pigs to irreversibly destroy target cells within a narrow range while leaving neighboring cells unaffected, and thus represents a promising new treatment for cancer, heart disease and other disease states that require removal of tissue. [23] Irreversible electroporation (IRE) has since proven effective in treating human cancer, with surgeons at Johns Hopkins and other institutions now using the technology to treat pancreatic cancer previously thought to be unresectable. [24]

Also first phase I clinical trial of gene electrotransfer in patients with metastatic melanoma was reported. [25] [26] Electroporation mediated delivery of a plasmid coding gene for interleukin-12 (pIL-12) was performed and safety, tolerability and therapeutic effect were monitored. Study concluded, that gene electrotransfer with pIL-12 is safe and well tolerated. In addition partial or complete response was observed also in distant non treated metastases, suggesting the systemic treatment effect. Based on these results they are already planning to move to Phase II clinical study. There are currently several ongoing clinical studies of gene electrotransfer, [27] where safety, tolerability and effectiveness of immunization with DNA vaccine, which is administered by the electric pulses is monitored.

Although the method is not systemic, but strictly local one, it is still the most efficient non-viral strategy for gene delivery.

N-TIRE Edit

A recent technique called non-thermal irreversible electroporation (N-TIRE) has proven successful in treating many different types of tumors and other unwanted tissue. This procedure is done using small electrodes (about 1mm in diameter), placed either inside or surrounding the target tissue to apply short, repetitive bursts of electricity at a predetermined voltage and frequency. These bursts of electricity increase the resting transmembrane potential (TMP), so that nanopores form in the plasma membrane. When the electricity applied to the tissue is above the electric field threshold of the target tissue, the cells become permanently permeable from the formation of nanopores. As a result, the cells are unable to repair the damage and die due to a loss of homeostasis. [28] N-TIRE is unique to other tumor ablation techniques in that it does not create thermal damage to the tissue around it.

Reversible electroporation Edit

Contrastingly, reversible electroporation occurs when the electricity applied with the electrodes is below the electric field threshold of the target tissue. Because the electricity applied is below the cells' threshold, it allows the cells to repair their phospholipid bilayer and continue on with their normal cell functions. Reversible electroporation is typically done with treatments that involve getting a drug or gene (or other molecule that is not normally permeable to the cell membrane) into the cell. Not all tissue has the same electric field threshold therefore careful calculations need to be made prior to a treatment to ensure safety and efficacy. [29]

One major advantage of using N-TIRE is that, when done correctly according to careful calculations, it only affects the target tissue. Proteins, the extracellular matrix, and critical structures such as blood vessels and nerves are all unaffected and left healthy by this treatment. This allows for a quicker recovery, and facilitates a more rapid replacement of dead tumor cells with healthy cells. [30]

Before doing the procedure, scientists must carefully calculate exactly what needs to be done and treat each patient on an individual case-by-case basis. To do this, imaging technology such as CT scans and MRI's are commonly used to create a 3D image of the tumor. From this information, they can approximate the volume of the tumor and decide on the best course of action including the insertion site of electrodes, the angle they are inserted in, the voltage needed, and more, using software technology. Often, a CT machine will be used to help with the placement of electrodes during the procedure, particularly when the electrodes are being used to treat tumors in the brain. [31]

The entire procedure is very quick, typically taking about five minutes. The success rate of these procedures is high [32] and is very promising for future treatment in humans. One disadvantage to using N-TIRE is that the electricity delivered from the electrodes can stimulate muscle cells to contract, which could have lethal consequences depending on the situation. Therefore, a paralytic agent must be used when performing the procedure. The paralytic agents that have been used in such research are successful [ citation needed ] however, there is always some risk, albeit slight, when using anesthetics.

H-FIRE Edit

A more recent technique has been developed called high-frequency irreversible electroporation (H-FIRE). This technique uses electrodes to apply bipolar bursts of electricity at a high frequency, as opposed to unipolar bursts of electricity at a low frequency. This type of procedure has the same tumor ablation success as N-TIRE. However, it has one distinct advantage, H-FIRE does not cause muscle contraction in the patient and therefore there is no need for a paralytic agent. [33] Furthermore, H-FIRE has been demonstrated to produce more predicable ablations due to the lesser difference in the electrical properties of tissues at higher frequencies. [34]

Drug and gene delivery Edit

Electroporation can also be used to help deliver drugs or genes into the cell by applying short and intense electric pulses that transiently permeabilize cell membrane, thus allowing transport of molecules otherwise not transported through a cellular membrane. This procedure is referred to as electrochemotherapy when the molecules to be transported are chemotherapeutic agents or gene electrotransfer when the molecule to be transported is DNA. Scientists from Karolinska Institutet and the University of Oxford use electroporation of exosomes to deliver siRNAs, antisense oligonucleotides, chemotherapeutic agents and proteins specifically to neurons after inject them systemically (in blood). Because these exosomes are able to cross the blood brain barrier, this protocol could solve the problem of poor delivery of medications to the central nervous system, and potentially treat Alzheimer's disease, Parkinson's disease, and brain cancer, among other conditions. [35]

Bacterial transformation is generally the easiest way to make large amounts of a particular protein needed for biotechnology purposes or in medicine. Since gene electrotransfer is very simple, rapid and highly effective technique it first became very convenient replacement for other transformation procedures. [36]

Recent research has shown that shock waves could be used for pre-treating the cell membrane prior to electroporation. [37] [38] This synergistic strategy has shown to reduce external voltage requirement and create larger pores. Also application of shock waves allow scope to target desired membrane site. This procedure allows to control the size of the pore.

Electroporation allows cellular introduction of large highly charged molecules such as DNA which would never passively diffuse across the hydrophobic bilayer core. [2] This phenomenon indicates that the mechanism is the creation of nm-scale water-filled holes in the membrane. [39] Electropores were optically imaged in lipid bilayer models like droplet interface bilayers [40] and giant unilamellar vesicles, [41] while addition of cytoskeletal proteins such as actin networks to the giant unilamellar vesicles seem to prevent the formation of visible electropores. [42] Experimental evidences for actin networks in regulating the cell membrane permeability has also emerged. [43] Although electroporation and dielectric breakdown both result from application of an electric field, the mechanisms involved are fundamentally different. In dielectric breakdown the barrier material is ionized, creating a conductive pathway. The material alteration is thus chemical in nature. In contrast, during electroporation the lipid molecules are not chemically altered but simply shift position, opening up a pore which acts as the conductive pathway through the bilayer as it is filled with water.

Electroporation is a dynamic phenomenon that depends on the local transmembrane voltage at each point on the cell membrane. It is generally accepted that for a given pulse duration and shape, a specific transmembrane voltage threshold exists for the manifestation of the electroporation phenomenon (from 0.5 V to 1 V). This leads to the definition of an electric field magnitude threshold for electroporation (Eth). That is, only the cells within areas where E≧Eth are electroporated. If a second threshold (Eir) is reached or surpassed, electroporation will compromise the viability of the cells, i.e., irreversible electroporation (IRE). [44]

Electroporation is a multi-step process with several distinct phases. [45] First, a short electrical pulse must be applied. Typical parameters would be 300–400 mV for < 1 ms across the membrane (note- the voltages used in cell experiments are typically much larger because they are being applied across large distances to the bulk solution so the resulting field across the actual membrane is only a small fraction of the applied bias). Upon application of this potential the membrane charges like a capacitor through the migration of ions from the surrounding solution. Once the critical field is achieved there is a rapid localized rearrangement in lipid morphology. The resulting structure is believed to be a "pre-pore" since it is not electrically conductive but leads rapidly to the creation of a conductive pore. [46] Evidence for the existence of such pre-pores comes mostly from the "flickering" of pores, which suggests a transition between conductive and insulating states. [47] It has been suggested that these pre-pores are small (

3 Å) hydrophobic defects. If this theory is correct, then the transition to a conductive state could be explained by a rearrangement at the pore edge, in which the lipid heads fold over to create a hydrophilic interface. Finally, these conductive pores can either heal, resealing the bilayer or expand, eventually rupturing it. The resultant fate depends on whether the critical defect size was exceeded [48] which in turn depends on the applied field, local mechanical stress and bilayer edge energy.

Gene electroporation Edit

Application of electric pulses of sufficient strength to the cell causes an increase in the trans-membrane potential difference, which provokes the membrane destabilization. Cell membrane permeability is increased and otherwise nonpermeant molecules enter the cell. [49] [50] Although the mechanisms of gene electrotransfer are not yet fully understood, it was shown that the introduction of DNA only occurs in the part of the membrane facing the cathode and that several steps are needed for successful transfection: electrophoretic migration of DNA towards the cell, DNA insertion into the membrane, translocation across the membrane, migration of DNA towards the nucleus, transfer of DNA across the nuclear envelope and finally gene expression. [51] There are a number of factors that can influence the efficiency of gene electrotransfer, such as: temperature, parameters of electric pulses, DNA concentration, electroporation buffer used, cell size and the ability of cells to express transfected genes. [52] In in vivo gene electrotransfer also DNA diffusion through extracellular matrix, properties of tissue and overall tissue conductivity are crucial. [53]

In the 1960s, it was known that by applying an external electric field, a large membrane potential at the two pole of a cell can be created. In the 1970s, it was discovered that when a membrane potential reached a critical level, the membrane would break down and that it could recover. [54] By the 1980s, this opening was being used to introduce various of materials/molecules into the cells. [55]


Could mRNA vaccines permanently alter DNA? Recent science suggests they might

April 9, 2021 (Children&rsquos Health Defense) &mdash Over the past year, it would be all but impossible for Americans not to notice the media&rsquos decision to make vaccines the dominant COVID narrative, rushing to do so even before any coronavirus-attributed deaths occurred.

The media&rsquos slanted coverage has provided a particularly fruitful public relations boost for messenger RNA (mRNA) vaccines &mdash decades in the making but never approved for human use &mdash helping to usher the experimental technology closer to the regulatory finish line.

Under ordinary circumstances, the body makes (&ldquotranscribes&rdquo) mRNA from the DNA in a cell&rsquos nucleus. The mRNA then travels out of the nucleus into the cytoplasm, where it provides instructions about which proteins to make.

By comparison, mRNA vaccines send their chemically synthesized mRNA payload (bundled with spike protein-manufacturing instructions) directly into the cytoplasm.

According to the Centers for Disease Control and Prevention (CDC) and most mRNA vaccine scientists, the buck then stops there &mdash mRNA vaccines &ldquodo not affect or interact with our DNA in any way,&rdquo the CDC says. The CDC asserts first, that the mRNA cannot enter the cell&rsquos nucleus (where DNA resides), and second, that the cell &mdash Mission-Impossible-style &mdash &ldquogets rid of the mRNA soon after it is finished using the instructions.&rdquo

A December preprint about SARS-CoV-2, by scientists at Harvard and Massachusetts Institute of Technology (MIT), produced findings about wild coronavirus that raise questions about how viral RNA operates.

The scientists conducted the analysis because they were &ldquopuzzled by the fact that there is a respectable number of people who are testing positive for COVID-19 by PCR long after the infection was gone.&rdquo

Their key findings were as follows: SARS-CoV-2 RNAs &ldquocan be reverse transcribed in human cells,&rdquo &ldquothese DNA sequences can be integrated into the cell genome and subsequently be transcribed&rdquo (a phenomenon called &ldquoretro-integration&rdquo) &mdash and there are viable cellular pathways to explain how this happens.

According to Ph.D. biochemist and molecular biologist Dr. Doug Corrigan, these important findings (which run contrary to &ldquocurrent biological dogma&rdquo) belong to the category of &ldquoThings We Were Absolutely and Unequivocally Certain Couldn&rsquot Happen Which Actually Happened.&rdquo

The findings of the Harvard and MIT researchers also put the CDC&rsquos assumptions about mRNA vaccines on shakier ground, according to Corrigan. In fact, a month before the Harvard-MIT preprint appeared, Corrigan had already written a blog outlining possible mechanisms and pathways whereby mRNA vaccines could produce the identical phenomenon.

In a second blog post, written after the preprint came out, Corrigan emphasized that the Harvard-MIT findings about coronavirus RNA have major implications for mRNA vaccines &mdash a fact he describes as &ldquothe big elephant in the room.&rdquo While not claiming that vaccine RNA will necessarily behave in the same way as coronavirus RNA &mdash that is, permanently altering genomic DNA &mdash Corrigan believes that the possibility exists and deserves close scrutiny.

In Corrigan&rsquos view, the preprint&rsquos contribution is that it &ldquovalidates that this is at least plausible, and most likely probable.&rdquo

Reverse transcription

As the phrase &ldquoreverse transcription&rdquo implies, the DNA-to-mRNA pathway is not always a one-way street. Enzymes called reverse transcriptases can also convert RNA into DNA, allowing the latter to be integrated into the DNA in the cell nucleus.

Nor is reverse transcription uncommon. Geneticists report that &ldquoOver 40% of mammalian genomes comprise the products of reverse transcription.&rdquo

The preliminary evidence cited by the Harvard-MIT researchers indicates that endogenous reverse transcriptase enzymes may facilitate reverse transcription of coronavirus RNAs and trigger their integration into the human genome.

The authors suggest that while the clinical consequences require further study, detrimental effects are a distinct possibility and &mdash depending on the integrated viral fragments&rsquo &ldquoinsertion sites in the human genome&rdquo and an individual&rsquos underlying health status &mdash could include &ldquoa more severe immune response &hellip such as a &lsquocytokine storm&rsquo or auto-immune reactions.&rdquo

In 2012, a study suggested that viral genome integration could &ldquolead to drastic consequences for the host cell, including gene disruption, insertional mutagenesis and cell death.&rdquo

Corrigan makes a point of saying that the pathways hypothesized to facilitate retro-integration of viral &mdash or vaccine &mdash RNA into DNA &ldquoare not unknown to people who understand molecular biology at a deeper level.&rdquo

Even so, the preprint&rsquos discussion of reverse transcription and genome integration elicited a maelstrom of negative comments from readers unwilling to rethink biological dogma, some of whom even advocated for retraction (though preprints are, by definition, unpublished) on the grounds that &ldquoconspiracy theorists &hellip will take this paper to &lsquoproof&rsquo that mRNA vaccines can in fact alter your genetic code.&rdquo

More thoughtful readers agreed with Corrigan that the paper raises important questions. For example, one reader stated that confirmatory evidence is lacking &ldquoto show that the spike protein only is expressed for a short amount of time (say 1-3 days) after vaccination,&rdquo adding, &ldquoWe think that this is the case, but there is no evidence for that.&rdquo

In fact, just how long the vaccines&rsquo synthetic mRNA &mdash and thus the instructions for cells to keep manufacturing spike protein &mdash persist inside the cells is an open question.

Ordinarily, RNA is a &ldquonotoriously fragile&rdquo and unstable molecule. According to scientists, &ldquothis fragility is true of the mRNA of any living thing, whether it belongs to a plant, bacteria, virus or human.&rdquo

But the synthetic mRNA in the COVID vaccines is a different story. In fact, the step that ultimately allowed scientists and vaccine manufacturers to resolve their decades-long mRNA vaccine impasse was when they figured out how to chemically modify mRNA to increase its stability and longevity &mdash in other words, produce RNA &ldquothat hangs around in the cell much longer than viral RNA, or even RNA that our cell normally produces for normal protein production.&rdquo

It is anyone&rsquos guess what the synthetic mRNA is doing while it is &ldquohanging around,&rdquo but Corrigan speculates that its enhanced longevity raises the probability of it &ldquobeing converted over into DNA.&rdquo

Moreover, because the vaccine mRNA is also engineered to be more efficient at being translated into protein, &ldquonegative effects could be more frequent and more pronounced with the vaccine when compared to the natural virus.&rdquo

Dollar signs

Corrigan acknowledges that some people may dismiss his warnings, saying &ldquoIf the virus is able to accomplish this, then why should I care if the vaccine does the same thing?&rdquo

He has a ready and compelling response:

&ldquo[T]here&rsquos a big difference between the scenario where people randomly, and unwittingly, have their genetics monkeyed with because they were exposed to the coronavirus, and the scenario where we willfully vaccinate billions of people while telling them this isn&rsquot happening.&rdquo

Unfortunately, the prevailing attitude seems to be that the &ldquorace to get the public vaccinated&rdquo justifies taking these extra risks.

In mid-November, after the Jerusalem Post told readers that &ldquowhen the world begins inoculating itself with these completely new and revolutionary vaccines, it will know virtually nothing about their long-term effects,&rdquo an Israeli hospital director argued that it&rsquos not worth waiting two more years to ferret out mRNA vaccines&rsquo &ldquounique and unknown risks&rdquo or potential long-term effects.

In the U.S., enthusiasm for mRNA technology is similarly unfettered. Just a few days after the CDC released updated data showing that more than 2,200 deaths of individuals who had received either the Pfizer or Moderna mRNA vaccines had been reported as of Mar. 26 , The Atlantic praised the technology, suggesting that the &ldquoingenious&rdquo synthetic mRNA technology behind Pfizer&rsquos and Moderna&rsquos COVID vaccines represented a &ldquobreakthrough&rdquo that could &ldquochange the world.&rdquo

Rather than dismiss the prospect of retro-integration of foreign DNA as a &ldquoconspiracy theory,&rdquo scientists should be conducting studies with the mRNA-vaccinated to assess actual risks.

For example, Corrigan believes that while in vitro data in human cell lines (one of the data sources examined by the Harvard-MIT researchers) offer &ldquoair tight&rdquo results, there is still a need to conclusively demonstrate real-life genomic alteration through &ldquoPCR, DNA sequencing or Southern Blot &hellip on purified genomic DNA of COVID-19 patients&rdquo &mdash and vaccinated individuals.

Yet instead of addressing these research gaps, companies are salivating over the potential to use human-edited mRNA to &ldquocommandeer our cellular machinery&rdquo and &ldquomake just about any protein under the sun.&rdquo

A March 10 press release pronouncing mRNA vaccines the clear winners of the COVID-19 vaccine race noted that all major pharmaceutical companies are now &ldquotesting out the [mRNA] technology by entering into license agreements and/or collaboration with well-established RNA companies.&rdquo

In old Disney cartoons, viewers often witnessed Donald Duck&rsquos rich uncle, Scrooge McDuck&rsquos, &ldquobulging eyes [turn] into oversized Vegas slot machine dollar signs&rdquo when contemplating opportunities to increase his already immense wealth.

Judging by pharmaceutical company executives&rsquo willingness to overlook mRNA vaccines&rsquo long-term &mdash and possibly multigenerational &mdash risks, they must be similarly entranced by dollar-sign visions of a never-ending pipeline of &ldquoplug and play&rdquo mRNA products.

LifeSiteNews has produced an extensive COVID-19 vaccines resources page. View it here.

© April 8, 2021 Children&rsquos Health Defense, Inc. This work is reproduced and distributed with the permission of Children&rsquos Health Defense, Inc. Want to learn more from Children&rsquos Health Defense? Sign up for free news and updates from Robert F. Kennedy, Jr. and the Children&rsquos Health Defense. Your donation will help to support us in our efforts.


Laboratory-designed plasmids contain a small number of genes that help transformation . These include:

  • An origin of replication . This is the specific sequence of nucleotides where DNA replication begins.
  • A multiple cloning site. This site contains recognition sites for specific restriction enzymes. These restriction enzymes can be used to ‘cut’ the plasmid so foreign DNA can be ‘pasted’ in by ligation .
  • A resistance gene . This gene codes for a protein the bacteria need in order to survive in a particular growth medium , for example, when a specific antibiotic is present.

Cloning DNA into a vector, step by step

To introduce foreign DNA into a circular vector, scientists carry out a three-step process:

Scientists first remove their gene of interest from the DNA sequences on either side of it. They can use restriction enzymes to do the cutting. These enzymes, which came originally from bacteria, cut DNA at specific sites in the sequence. If there’s not enough DNA for successful cutting or no suitable restriction enzyme recognition sites around the gene, scientists first use polymerase chain reaction (PCR) to make many more copies. By designing their PCR primers carefully, they can introduce new restriction sites on either side of the copied DNA sequence.

Opening up the vector

Next, scientists make a cut in the circular DNA sequence of the vector. They use the same restriction enzymes as they used to cut out the gene in step 1. This turns the vector into a linear molecule and makes it ready to accept the new piece of DNA.

Sticking the vector and the gene together

The final step in cloning is to incorporate the DNA of interest into the vector. Scientists mix the gene and the opened vector together with a bacterial enzyme called DNA ligase . The ligase sticks DNA ends together to form a single circular molecule that includes both the vector and the gene.

Find out more in these articles:


Human Aborted Fetal Cell DNA in Vaccines.

Take a look at the vaccine excipient and media summary compiled by the CDC (this is where you will find the ingredients and other biological and chemical materials used in the production of vaccines).

Much of what is listed in the table is not self-explanatory and requires additional research and investigation. Ultimately, these terms relate to chemical substances, antibiotics, and human or animal cells and serums (a fraction of blood) used to manufacture each vaccine. While they are not labeled as “ingredients”, residual amounts of the excipients and media end up in the vaccines your child receives at the doctor’s office, and are therefore also unintended ingredients and/or known contaminants.

An “excipient” is an additive which helps to stabilize and/or enhance the therapeutic activity of active ingredients in a drug or vaccine. “Media” refers to growth mediums or cell cultures, which is a mixture of ingredients, in which vaccine antigens are grown.

Aborted fetal cells.

When you read “cell culture materials” in the image above, this is where cells from aborted fetuses/unborn children factor into the manufacturing process of vaccines. Where’s the evidence for this?

“There are two particular fetal cell lines that have been heavily used in vaccine development. They are named according to the laboratory facilities where they were developed. One cell line is known as WI-38, developed at the Wistar Institute in Philadelphia, PA. The other is MRC-5, developed for the Medical Research Council in England.”

– Right to Life, Life Notes: Vaccines, Abortion, & Fetal Tissue.

Take a look at this informative article by ProCon.org which provides descriptions of some of the vaccine excipients and media. Then go to the MMR vaccine. Under the heading titled, “Gross Mediums and Process Ingredients”, you’ll find “WI-38 human diploid lung fibroblasts”.

From the link, this is the description of “WI-38”:

“Winstar Institute 38, human diploid lung fibroblasts derived from the lung tissues of a female fetus aborted because the family felt they had too many children in 1964 in the United States.”

The WI-38 fetus was aborted at three months (or about 14 weeks) gestation. These cells are even available for purchase, here.

Now let’s go to the Pentacel vaccine (DTaP, Polio, Hib). Here, alongside aluminum phosphate, formaldehyde, polysorbate 80, and more, you’ll find “MRC-5 cells“. Here’s the description:

“Medical Research Council 5, human diploid cells (cells containing two sets of chromosomes) derived from the normal lung tissues of a 14-week-old male fetus aborted for “psychiatric reasons” in 1966 in the United Kingdom, Eagle’s Basal Medium in Earle’s balanced salt solution with bovine serum.”

Not just two abortions.

At this point, defenders of vaccines will state that these cell lines were taken from just two fetuses, which were aborted many years ago.

Unfortunately, there is evidence that over 80 fetuses were aborted for the research and development of the rubella vaccine alone:

“The rubella virus clinically named RA273 (R=Rubella, A=Abortus, 27=27th fetus, 3=3rd tissue explant) was then cultivated on the WI-38 aborted fetal cell line. A later research paper by Stanley Plotkin would reveal that 40 more babies were aborted after RA273 was successfully isolated, with virus strains taken from 34 of them.[13A] This means a total of over 80 separate, elective abortions recorded were involved in the research and final production of the present day rubella vaccine: 21 from the original WI-1 through WI-26 fetal cell lines that failed, plus WI-38 itself, plus 67 from the attempts to isolate the rubella virus.”

New cell lines.

Due to the fact that WI-38 and MRC-5 cannot be used indefinitely, new cell lines are being developed. This means that new elective abortions are currently taking place for the continued production of more and more vaccines.

From the study linked above, published in 2015:

“We obtained 9 fetuses through rigorous screening based on carefully specified inclusion criteria… Walvax-2 was derived from a fetal lung tissue, similar to WI-38 and MRC-5, and was obtained from a 3-month old female fetus aborted because of the presence of a uterine scar from a previous caesarean birth by a 27-year old healthy woman.”

“The tissues from the freshly aborted fetuses were immediately sent to the laboratory for the preparation of the cells.”

Residual DNA fragments in vaccines.

Apart from any moral conflict one might have over the use of aborted fetuses for vaccine production, we need to remember that DNA from the aborted fetuses actually ends up in vaccines as a contaminant.

“The issue of concern is that many common vaccines were developed using cell lines that originally were cells taken from electively aborted babies. The vaccines themselves do not contain fetal cells, but there are significant “residual” biological components from the fetal cells that have been assimilated into the vaccine, including cell proteins and measurable portions of fetal DNA.”

Independent research has found that vaccines manufactured in human fetal cell lines contain “unacceptably high levels of fetal DNA fragment contaminants”. These fragments, while in trace amounts, are still biologically active once injected into the body of another individual via vaccine. Vaccines elicit systemic immune activation and inflammatory responses, which increases the likelihood of foreign DNA uptake into the host’s genome. And in fact, it has been found that fetal cell DNA can spontaneously integrate into the genome of the vaccinated person.

In addition to this, a disturbing correlation has been found between the use of aborted fetal cell produced vaccines and sudden increase in the rate of autistic spectrum disorder.

“In 1979, coincident with the first autism disorder change point, vaccine manufacturing changes introduced human fetal DNA fragments and retroviral contaminants into childhood vaccines (Victoria et al., 2010). While we do not know the causal mechanism behind these new vaccine contaminants and autistic disorder, human fetal DNA fragments are inducers of autoimmune reactions, while both DNA fragments and retroviruses are known to potentiate genomic insertions and mutations (Yolkenetal.,2000 Kurth1998 USFood and Drug Administration 2011).”

What we know.

For a more comprehensive list, visit Cogforlife.org.

Human babies have been and are still being sacrificed for use in the research and development of vaccines. DNA from these aborted fetuses is present in vaccines, and independent scientific research has found that there is the potential for insertional mutagenesis to occur within the vaccinated person. Unfortunately, vaccine manufacturers do not test their products for mutagenic potential. The long term adverse effects of using aborted fetal cells for vaccine production are virtually unknown, yet millions of these vaccines are administered every day.

Screen shot of the Pentacel vaccine package insert.

*The ultrasound image used for this article is the image of my daughter at 14-weeks gestation.