Ruppy the Glow-in-the-dark Puppy

Meera Singhal
11 min readApr 4, 2021

Look at this.

You must be thinking — AWW that's adorable!! Theres not much that can make this fur-ball much cuter in my eyes. However, some scientists thought differently. Through scrazy sciences like gene editing, CRISPR, transgenic animals, etc. scientists were able to make a beagle just like this one, glow in the dark!

WOAH! Meera there is no way your telling me that scientist figured out how to make puppies even cuter!?

Thats right. They did. And through this article Ill be talking about how they did this crazy thing!

I threw a couple of fancy terms like gene editing and CRISPR but what exactly do they mean? (If you know what they mean, skip this basic lesson in gene editing and head into the spicy content) If you don’t, stick around for this part, it is important!

Detailed Image of a DNA strand with its bases

First things first, let us talk about DNA. DNA stands for deoxyribonucleic acid. DNA is made up of nucleotides, or biological building blocks! DNA is one of the most important molecules for not only humans and animals, but also for most other organisms as well. In essence, DNA contains our genes and hereditary materials- its what makes us unique! DNA is constructed by molecules, each of which consists of two strands that are wound around each other while each stand is held together by bonds between the bases. Each strand is coded with four-letter alphabet- A, T, C, and G. These letters form complementary base pairs: A only bonds with T, C only bonds with G.

Image of RNA versus DNA

Next, lets talk a bit more about RNA. RNA stands for ribonucleic acid. It is a complex compound of high molecular weight that functions in cellular protein synthesis and replaces DNA (deoxyribonucleic acid) as a carrier of genetic codes in some viruses. The nitrogenous bases in RNA are adenine, guanine, cytosine, and uracil, which replace the thymine in DNA. Mutations occur when the order of the genetic code is changed. Gene editing is essentially artificially inducing and specifying a mutation.

Before we jump into Ruppy, it would a good idea to go over CRISPR. In my mind, I like to think of CRISPR with an analogy. For example, take a working document. In a document, if I suspected that I misspelled a phrase or word, we can always use the find and replace bar to fix any errors. We can edit, delete add, basically just alter the words we wrote. Similarly, in our DNA, we have the same function. This function is taken on by a system called CRISPR Cas9.

CRISPR is short for;

Clustered

Regularly

Interspaced

Short

Palindromic

Repeats

CRISPR consist of two main components. The Cas9 protein has the ability to cut through DNA and a guide RNA that can recognize the sequence of the DNA that must be edited.

Image of Scientist extracting specific base pairs, just like CRISPR

To use CRISPR Cas9, scientists first identify the sequence of the human genome that is causing a health problem. Then they create a specific guide RNA to recognize the specific strands of A, C, G, and T’s in the DNA. The guide RNA is attached to the DNA cutting enzyme Cas9. After that, this complex is introduced to the target cells. It locates the target letter sequences and cuts the DNA. At that point, scientists can then edit the existing genome by modifying, deleting, or adding new sequences (just like a word document!). This process efficiently makes CRISPR Cas9 an efficient cut-and-paste tool for DNA editing. In the future, scientists hope to use CRISPR Cas9 to develop critical advances in patient care or even cure lifelong diseases.

Image of Gene targeting

There is one last thing that we should go over before I talk about Ruppy, genome sequencing. Genomic sequencing is a process for analyzing a sample of DNA taken from your blood. In the lab scientists and lab, techs extract DNA and prepare it for sequencing. Within every normal cell, there are 23 different pairs of chromosomes. Chromosomes are structuring that house DNA. DNA is coiled into a shape called the double helix. The double helix can be unwound into a ladder shape. This “ladder” is made out of paired chemical letters called bases. Overall, our DNA contains about 6 billion bases (per human!) To read the sequence of bases in DNA samples are inserted into a sequencing instrument where high-frequency sound waves break the DNA into smaller pieces that are only about 600 bases long. Special tags are added to the ends of the fragmented DNA. These tagged strands of DNA can then attach to a glass slide in a sequencer. Each piece of DNA has been copied hundreds of thousands of times which in turn creates clusters of identical DNA fragments. Next, the sequencer reads the DNA one base at a time using different colored tags for each DNA base special sensor within the machine to detect the different colored tags. This sequence of colors reveals the DNA sequence of each fragment. Powerful computers piece together these individual DNA fragments and reveal the sequence of your DNA then medical experts use specialized software to analyze and compare the DNA sequences. This helps identify the genetic variants and completes the process of sequencing.

An adorable picture of Ruppy in the dark.. and he's glowing!

Now that you know a brief overview of the biological structures like DNA/RNA, CRISPR, and genome editing, let's talked about Ruppy. Ruppy is a transgenic animal. A transgenic animal, or more commonly known as a genetically modified animal whose genome has been altered by the transfer of a gene or genes from another species or breed. Transgenic animals are crucial research and experiments for advancing medicine. These animals are constantly used in the lab as different models in biotechnical/medical research. Over 95 percent of the transgenic animals used in these trials are actually genetically modified rodents (especially mice). They are crucial tools for researching various human diseases, as well as they are being used to understand the gene function in the context of disease susceptibility, progression, and to determine response from a therapeutic intervention. Mice have also been genetically modified by scientists to naturally produce antibiotics for humans to be used as therapeutics. Seven out of the eleven monoclonal antibody drugs approved by the FDA between 2006 and 2011 were derived from transgenic mice.

Transgenic farm animals are also being explored as a means to produce large quantities of complex human proteins for the treatment of human disease. Such therapeutic proteins are currently produced in mammalian cell-based reactors, but this production process is expensive. In 2008, for example, the building of a new cell-based manufacturing facility for one therapeutic protein was estimated to cost over US$500 million. A cheaper option would be to develop a means to produce recombinant proteins in the milk, blood or eggs of transgenic animals. Progress in this area, however, has been slow to date. Only two biomedical products have so far received regulatory approval. The first is human antithrombin III, a therapeutic protein produced in the milk of transgenic goats, which is used to prevent clots in patients with hereditary antithrombin deficiency receiving surgery or undergoing childbirth. A relatively small herd of goats (about 80) can supply enough human antithrombin III for all of Europe. The second product is a recombinant human C12 esterase inhibitor produced in the milk of transgenic rabbits. This is used to treat hereditary angioedema, a rare genetic disorder that causes blood vessels in the blood to expand and cause skin swellings.

So why are transgenic animals important? As I previously mentioned, transgenic animals have helped advance biomedical research, been used to develop antibodies and therapeutic drugs, fighting human diseases, and more!

The ability to produce transgenic animals depends on a variety of different components. One of the first things needed to generate transgenic animals is the ability to transfer embryos. The first successful transfer of embryos was achieved by Walter Heape in Angora rabbits in 1891. Another important component is the ability to manipulate the embryo. In vitro manipulation of embryos in mice was first reported in the 1940s using a culture system. What is also vital is the ability to manipulate eggs.

Image Source

This was made possible through the efforts of Ralph Bruster, attached to the University of Pennsylvania, who in 1963 devised a reliable system to culture eggs, and that of Teh Ping Lin, based at the California School of Medicine, who in 1966 outlined a technique to micro-inject fertilized mouse eggs which enabled the accurate insertion of foreign DNA.

The first genetic modification of animals was reported in 1974 by the virologist Rudolph Jaenisch, then at the Salk Institute, and the mouse embryologist Beatrice Mintz at Fox Chase Cancer Center. They demonstrated the feasibility of modifying genes in mice by injecting the SV40 virus into early-stage mouse embryos. The resulting mice carried the modified gene in all their tissues. In 1976, Jaenisch reported that the Moloney Murine Leukemia Virus could also be passed on to offspring by infecting an embryo. Four years later, in 1980, Jon Gordon and George Scango together with Frank Ruddle, announced the birth of a mouse born with genetic material they had inserted into newly fertilized mouse eggs. By 1981 other scientists had reported the successful implantation of foreign DNA into mice, thereby altering the genetic makeup of the animals. This included Mintz with Tim Stewart and Erwin Wagner at the Fox Chase Cancer Center in Philadelphia; Brinster and Richard Palmiter at the University of Washington, Seattle; and Frank Costantini and Elizabeth Lacy at Oxford University.

Such work laid the basis for the creation of transgenic mice genetically modified to inherit particular forms of cancer. These mice were generated as a laboratory tool to better understand the onset and progression of cancer. The advantage of such mice is that they provide a model which closely mimics the human body. The mice not only provide a means to gain greater insight into cancer but also to test experimental drugs. This work also laid the basis for many more transgenic animals to come!

Usually, the DNA containing parts of the genres are the target, as well as a reporter gene, and a dominant selectable marker which is assembled in bacteria. Gene targeting methods are often established for several model organisms and they vary depending on the species that are used in the trials. To target genes in mice, the DNA is inserted into mice’s embryonic stem cells in culture. Cells with this insertion can contribute to a mice’s tissues through embryo injections. Finally, a specific type of mice, chimera mice, have modified cells that made up the reproductive organs, are bred for these experiments. After this step in the progress, the entire body of the test subject is based on the selection of embryonic stem cells.

How was Ruppy created? Ruppy was created through something as simple as a petri dish. Scientists hand grew in form a few cells into a dog. This procedure of artificial growth had been done before but never with the addition and altering of genes. They added the NPOE34 gene to a beagle with triggered another gene called the DFGR89 gene. This caused Ruppy, the glowing dog, to glow.

Beyeong-Chun Lee headed a team of scientists that created Ruppy, They first infected it with dog fibroblast cells combined with a virus that inserted the fluorescent gene into a cell’s nucleus. After that, they then transfer the fibroblast’s nucleus to another dog’s egg cells with its nucleus removed. After a few hours of dividing in tin a petri dish, researchers implanted the clone embryo into a surrogate mother to grow the glowing dog. Starting with 344 embryos implanted into 20 dogs, Lee’s team ended up with seven pregnancies. One fetus died about half way through term, while an 11-week-old puppy died of pneumonia after its mother accidentally bit its chest. Five dogs are alive, healthy and starting to spawn their own fluorescent puppies, Ko says.

To further understand this, try to think of it like an analogy. Think of ruppy like a glow stick. Before the glowsticks is bent, there's nothing special to it- its just like any other cylinder of plastic. Similarly, Ruppy is just like any other begale in broad daylight but when night time hits, BAM- nothings the same anymore. When you finally bend the glow stick, the toxins inside it activate at it glows. Simialrly, a gene in Ruppy can only activate when it is pitch black.

Besides the low efficiency of cloning — just 1.7 per cent of embryos came to term — another challenge to creating transgenic dogs is controlling where in the nuclear DNA a foreign gene lands. Lee’s team used a retrovirus to transfer the fluorescent gene to dog fibroblast cells, but they could not control where the virus inserted the gene. This would seem to prevent researchers from making dog “knockouts” lacking a specific gene or engineering dogs that produce mutant forms of a gene. These knockout procedures are now commonly done in mice and rats, and three researchers earned a Nobel prize in 2007 for developing this method, called “gene targeting”.

Gene Targeting

Gene targeting (as know as a replacement strategy) is a genetic technique that uses a form of gene editing called homologous recombination to modify a type of gene called an endogenous gene. This method is often used to delete genes, remove exons, adding different genes, modify individual base pairs, etc. Gene targeting can either be conditional or permanent. When gene targeting is conditional the conditions can vary from a specific time in the development of the organism, or it can limit it to a specific tissue. Gene targeting requires the creation of a specific vector for each gene of interest. However, it can be used for any gene, regardless of transcriptional activity or gene size.

Through this crazy process, Ruppy was created, and ever since, more transgenic animals have been born out of labs for different purposes and experiments. Zooming ahead 9 years, Ruppy has inspired so many different advances in biological sciences today. My favorite “spin-off” from ruppy is the inspiration of genetically engineering dog and transfusing genes into animals to potentially save their lives!

TL’DR

  • DNA/ RNA. The building blocks of life.
  • CRISPR. A new form of gene editing!
  • Genome Sequencing. Trageting genes.
  • Transgeneic animals. Animals that have been genetically engennered.
  • Ruppy! The star of this article + the jumpingstone for so many new technologies!

Hello! My name is Meera Singhal, and I am a 13 year old currently fascinated by the field of biotechnology, specifically stem cells and gene editing. I’ve written articles about biotechnology, mindset tips, and a whole variety of up-and-coming topics. Interested? Check out my medium, LinkedIn, YouTube, or TKS Life Portfolio for more content! Curious to see more about me? Consider subscribing to my Newsletter! Thank you so much!

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Meera Singhal

Biotech Enthusiast at TKS | Stem Cells | Gene Editing