HAMES_DBI.A12

 Project Proposal

    Hypothesis: By regulating the gene SLC5A1, we can also regulate the expression of the sodium-glucose cotransport 1 and the genetic disorder GGM. From this, we are able to induce GGM into a mouse through editing the gene. To test the expression of the mutation, we will feed the mouse food that contains glucose/galactose. If the mouse experiences diarrhea and excessive dehydration, we can conclude the gene editing as successful. 

    Introduction: For this semester, the digestive protein I chose all the way back in the beginning of February was sodium-glucose cotransport 1. Over the course of the semester, I learned a lot about this protein and the gene that is associated with it by traveling and mining information through different databases. Sodium-glucose cotransport 1 (SGLT1) is mainly found in kidney proximal tubes because it regulates sodium/sugar uptake levels in intestinal cells. The mutation in the gene that encodes for sodium-glucose cotransport 1 causes a recessive disease disorder called congenital galactose/glucose malabsorption (GGM). The mutation in the gene causes the enzyme driving the transport of glucose into the cells to not work properly, leading to excretion of sugars in the stool. "The protein product of SGLT1 then moves the glucose and the galactose from the lumen of the small intestine into intestinal cells. Usually the mutations carried by GGM individuals result in nonfunctional truncated SGLT1 proteins or in the improper placement of the proteins such that they can not transport glucose and galactose out of the intestinal lumen" (National Center for Biotechnology Information (US).This excretion of sugars in the stool forces lots of water to be wasted along with it, leading to massive amounts of dehydration and diarrhea. Though this metabolic disease infects people from birth and is likely fatal, it only infects around 300 people worldwide. In this project, I want to be able to regulate the SLC5A1 gene that encodes for SGLT1, and cause GGM through its mutation. I can regulate the expression of SLC5A1 through various techniques like RNA sequencing, microarrays, polymerase chain reaction (PCR), and CRISPR which I have learned throughout the semester. Through regulation of the gene expression, I hope to learn more about how its mutation can cause GGM and research possibly what procedures, medications, and ways we can use to treat/control it. In this project I propose that I mutate the SLC5A1 gene to induce GGM into an organism. Since the disorder is very rare and hard to get ahold of, it is more practical to induce GGM rather than finding an organism with the disorder and curing it. 

    Methods: This research project will utilize 5 databases and the techniques mentioned in the introduction to learn more about how the SLC5A1 codes for sodium-glucose cotransport 1 and how the gene's mutation leads to GGM in an organism. First, we can use the Reactome and UniProt databases to understand more about the gene's role in metabolic digestive pathways, and what biomolecules are used along with it. For example, we are able to see in the Reactome database that different solute carriers and other genes are used in conjunction with SLC5A1 to help transport sugars such as glucose, galactose, fructose, and mannose. We are also able to find the specific variants in the DNA sequencing from the UniProt database as well as more specific information on the protein/gene. From there, we need to find any other organisms that have the gene/protein present besides human, since it is more practical and ethical to obtain an organism for research other than human. Using the NCBI database, we are able to see that many animals, mostly mammals, have the gene. Many mammals include monkeys, mice, frogs, bats, owls, fish, zebras, etc. We could use any of these but I propose that we use Mus musculus (house mice) because rats and mice are usually used for research experiments due to their resemblance to humans and ability to reproduce rather quickly. Then, we can use the ORegAnno and VarSite databases to learn more about transcription factors/elements of the gene and protein to further learn more about regulation. We can also use UCSC Genome Browser to find the frequency of SINES and microsatellites on the gene in order to track mutations. Once we obtain the experimental house mice, we can take out a blood sample and isolate the gene SLC5A1 from RNA sequencing and microarrays of the house mice. From there we can use PCR to replicate and amplify the gene's region. By copying this section of the DNA, we can make it easier to introduce a mutation as well as we can use gel electrophoresis to measure the RNA sequencing before and after the mutation. After this we can use the CRISPR technology (ex vivo in the second image below) to excise the specific gene SLC5A1, modify it to introduce the mutation, and the reintroduce it in the mice. CRISPR is a new and amazing tool that'll help edit the genome of the mice and introduce the mutation into the specific gene that we want, SLC5A1. Many research labs and experiments, like in this proposed project, are utilizing CRISPR to its fullest extent - "A growing number of preclinical studies based on rodent and other animal models indicate that CRISPR-Cas systems have the potential for therapeutic usage in different diseases, including genetic diseases, infectious diseases, cancers, immunological diseases (autoimmunity and immunodeficiency), etc...The CRISPR-Cas tools are being widely used to correct genetic variants with the hope of treating many human genetic diseases, such as inherited blood disorders (sickle cell disease, β-thalassemia, and hemophilia)..."(Liu et. al., 2021). We can then take a sample of newly mutated sequence, perform PCR, and put it through gel electrophoresis in order to compare it to the control, the normal RNA sequence. After reintroduction and replication of the newly mutated genome sequence in the house mice, we can test the expression of the mutation to see if the autosomal recessive disorder GGM develops in the mice by feeding them sugar and observing their reactions. GGM does not allow for the absorption of the glucose and galactose in the intestinal tissues, making it not possible for the mice to digest the sugar if the expression of the gene does occur in the mice. 

Image provided via Rodriguez et. al. (2019). 

Image provided via Liu et. al (2021).

    Timeline: 

1. Research the metabolic pathways of SLC5A1 and SGLT1 extensively- 1 week

2. Obtain Mus musculus (sample house mice)- amount of time varies

3. Research potential variations, transcription factors, SINES, and microsatellites- 2 weeks

4. Isolate SLC5A1 from mouse blood sample using RNA sequencing and microarrays- 4 days

5. Perform PCR and then gel electrophoresis- 1-2 days

6. Edit and induce mutation variant into SLC5A1- time varies

7. Perform PCR and then gel electrophoresis of mutated gene sequence and compare to control- 1-3 days

8. Reintroduce mutation into mice- time varies

9. Feed mice and observe expression of gene mutation (GGM)- time varies

    Citations: 

    Liu, W., Li, L., Jiang, J., Wu, M., & Lin, P. (2021). Applications and challenges of CRISPR-Cas gene-editing to disease treatment in clinics. Precision clinical medicine, 4(3), 179–191. https://doi.org/10.1093/pcmedi/pbab014

    National Center for Biotechnology Information (US). Genes and Disease [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 1998-. Glucose galactose malabsorption. Available from: https://www.ncbi.nlm.nih.gov/books/NBK22210/

    Rodríguez-Rodríguez, D. R., Ramírez-Solís, R., Garza-Elizondo, M. A., Garza-Rodríguez, M. L., & Barrera-Saldaña, H. A. (2019). Genome editing: A perspective on the application of CRISPR/Cas9 to study human diseases (Review). International journal of molecular medicine, 43(4), 1559–1574. https://doi.org/10.3892/ijmm.2019.4112