The new method facilitates genetic studies of intestinal epithelial function and disease

by Kerstin Wagner, Leibniz-Institut für Alternsforschung – Fritz-Lipmann-Institut eV (FLI)

The new method facilitates genetic studies of intestinal epithelial function and disease

Lentiviral injection of E8.0 mouse embryos achieves stable targeting of the intestinal epithelium in adult mice. Credit: BMC Biology (2023). DOI: 10.1186/s12915-022-01466-1

With a length of about eight meters and its innumerable finger-shaped protrusions, called villi, the intestine represents the largest contact surface within our body that comes into contact with ingested food.

As the most important organ of the digestive system, its main tasks include the absorption of nutrients from food, the supply of energy and the excretion of harmful metabolites. The intestinal barrier acts as a kind of protective wall which prevents germs or foreign substances from entering the body and thus has a decisive influence on health (immune defences).

Part of the intestinal wall is the intestinal lining (intestinal epithelium), which lines the inside of the intestine and plays an important role in the absorption of water, electrolytes, and nutrients. It is also subject to a continuous renewal process and is the fastest self-renewing tissue in adult mammals, with a regeneration time of 3-10 days.

Despite the diverse functions of the intestine and the enormous importance for health, little is known so far – despite intensive research – about which genes play an important role in digestion or in the development of intestinal diseases.

Researchers at the Leibniz Institute on Aging—Fritz Lipmann Institute (FLI) in Jena, Germany, in collaboration with partners at the Fred Hutchinson Cancer Center in Seattle, USA, have now achieved an important milestone: They have developed a new method that on the one hand it allows genetic studies for all regions of the intestine and on the other hand it can be used to study in more detail the influence of genes on carcinogenesis, the aging process and host-microbiome interactions.

The current research findings have now been published in the journal BMC Biology.

Mutation analysis of a single gene versus screening a large number of genes

“Using special germline mutations, it is currently possible to deactivate individual genes in the intestinal epithelium and investigate their influence. But despite many efforts, there is still no method that can be used to study a large number of genes simultaneously,” he explains K. Lenhard Rudolph, research group leader at FLI and professor of molecular medicine at FSU Jena. But such an approach would be necessary, because the human genome has over 25,000 genes, about three-quarters of which are active in our gut.

‘Furthermore, researching the function of these genes by targeted single-gene mutation analyzes is very expensive and also time-consuming,’ adds Prof. Rudolph. In principle, it is possible to induce mutations in individual cells of a tissue by using virus particles containing specific gene sequences. With this approach, screening studies on a large number of genes can be performed simultaneously.

“This method is already used today for the examination of skin, liver and blood stem cells, but so far it has not been transferable to the intestinal epithelium,” says the stem cell researcher, “because the stem cells of the epithelium bowel are deeply hidden in the crypts (the deep sacs between the villi) and are therefore very difficult to access for gene transfer by means of viral particles.”

Disadvantages of previous research methods

‘Currently known methods for studying the intestinal epithelium use intestinal organoids, tiny mini-organs grown in cell cultures and genetically modified with the addition of viruses,’ adds Dr. George B. Garside of FLI, lead author of the study.

“These modified mini-organs are then injected into the intestinal epithelium of immunocompromised mice, and the influence of the genes on intestinal function is then studied.”

However, this method has several weaknesses. Organoids must first be grown and propagated in cell culture in the laboratory before they can later be genetically modified by specific viral vectors. All of this takes place outside the organism, which means that the mini-organs lack their natural environment, which, however, regulates the natural development and maintenance of the intestinal epithelium.

Triggered by the unnatural growth conditions in the culture, changes can occur in intestinal stem cells, which therefore do not accurately reflect the function of genes under natural conditions.

Problems also arise during transplantation, because due to the length of the intestine and the poor accessibility from the outside, the transplantation of organoids remains limited to the lower colon and has an overall low efficiency, limiting the study to a few genes.

“Because of all these limitations and problems, it was necessary to find a new method that would allow the insertion of genetic modifications in situ, directly into the natural environment of the undisturbed intestinal epithelium, as well as allow the study of the function of multiple genes in the natural intestinal epithelium,” says Dr. Garside. ‘This also has advantages in that cells in culture can undergo abnormal selection and changes that perturb the identification of gene functions in unmodified cells.’

New method to study the intestinal epithelium

Researchers have successfully developed a robust and replicable procedure that makes it possible to introduce a large number of genetic modifications into stem cells of the unmodified natural intestinal epithelium without the need for transplantation. “We used laboratory mice for our study, which in many respects closely mirror the physiological conditions of the human intestinal epithelium,” reports Prof. Rudolph.

Due to the difficult to access anatomical location of the stem cells, which are deeply hidden in the crypts of the intestinal epithelium of adult mice, a special microinjection technique was used during the early embryonic development of the mice. This is because in this early stage of development, the intestine is still inside out, making it more accessible to the introduction of targeted genetic modifications.

Huge potential – a wide range of applications

The study results show that the new technique can be used to study gene function in the intestinal epithelium both during development and in adult animals. Furthermore, the method has the potential to identify genes that impact carcinogenesis, aging and interactions between the microbiome (the bacteria in the gut) and the host.

The main advantage of this method is that it allows in vivo genetic screens to be performed in the undisturbed intestinal epithelium of mice and possibly also in other model organs. Thus, all regions of the gastrointestinal tract, including the stomach, small intestine, and colon, can be genetically modified and studied.

What is also significant is that this technique can be applied to other endoderm-derived organs, such as the pancreas, liver, bladder, and lung, so that the method could be extremely useful in other research areas as well. . The development of this method is of great importance for the study of these organs and could particularly accelerate discoveries on the biology, function and development of intestinal epithelial diseases.

More information:
George B. Garside et al, In situ lentiviral targeting of stem cells in the unperturbed intestinal epithelium, BMC Biology (2023). DOI: 10.1186/s12915-022-01466-1

Provided by Leibniz-Institut für Alternsforschung – Fritz-Lipmann-Institut eV (FLI)

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