The new method makes genetic analyzes possible for all regions of the gut



With its length and many folds and protrusions, the intestine represents the largest contact surface between our body and the food taken in from the outside. However, little is currently known about the genes that play an important role in digestion or contact with foreign matter. Researchers from the Leibniz Institute on Aging – Fritz Lippmann Institute (FLI) in Jena, Germany, and Fred Hutchinson Cancer Center, Seattle, USA, have succeeded in developing a new method that makes genetic analyzes possible for all regions of the gut. This will facilitate future research into how genes affect the normal function of the gut lining, as well as their impact on disease and aging.

The intestine is about eight meters long and has the backs of countless finger-shaped projections, known as villi, representing the largest contact surface on the interior of the body that comes into contact with food that has been eaten. As the most important organ of the digestive system, its main functions include absorbing nutrients from food, providing energy, and excreting harmful metabolites. The intestinal barrier acts as a kind of protective wall that prevents germs or foreign substances from entering the body and thus has a decisive influence on health (immune defence).

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 also undergoes a continuous process of regeneration and is the fastest self-renewing tissue in adult mammals, with regeneration time ranging from 3 to 10 days. Despite the diversity of bowel functions and their enormous importance to health, little is known to date – despite extensive research – about which genes play an important role in digestion or in the development of intestinal diseases.

Researchers from the Leibniz Institute on Aging – Fritz Lippmann Institute (FLI) in Jena, Germany, in collaboration with partners from the Fred Hutchinson Cancer Center in Seattle, USA, have achieved an important achievement: they have developed a new method on one side that enables genetic studies of all regions of the intestine , and on the other hand can be used to investigate in more detail the influence of genes on carcinogenesis, the aging process, and host interactions with the microbiome. The results of the current research have now been published in the journal BMC Biology.

Single-gene mutation analysis versus screening for a large number of genes

Using germline-specific mutations, it is currently possible to turn off individual genes in the gut epithelium and investigate their effect. But despite many efforts, there is still no method that can be used to study a large number of genes simultaneously.”


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 contains more than 25,000 genes, about three-quarters of which are active in our gut.

“Moreover, research into the function of these genes through targeted single gene mutation analyses is very expensive and also very time consuming,” adds Professor Rudolph. In principle, it is possible to induce mutations in individual cells of a tissue using viral particles containing specific genetic sequences. Using this approach, screening studies can be performed on a large number of genes simultaneously. “This method is already used today to examine skin, liver and blood stem cells, but it has not been transferred to the intestinal epithelium until now,” the stem cell researcher confirms, “because stem cells in the intestinal epithelium are deep hidden in the crypts (deep pockets between the villi) and therefore difficult to access. to it for gene transfer by viral particles.

Disadvantages of previous research methods

Adds Dr. George B. These modified mini-organs are then injected into the intestinal epithelium of immunodeficient mice and the effect of genes on intestinal function is then studied.

However, this method has many weaknesses. Organoids must first be grown and propagated in in vitro cell culture before being subsequently genetically modified with specific viral vectors. All this happens outside the organism, which means that the microorganisms lack their natural environment, which, however, regulates the normal development and maintenance of the intestinal epithelium. Due to abnormal growth conditions in culture, changes can occur in intestinal stem cells, which then do not accurately reflect the function of genes under normal conditions.

Problems also arise during transplantation, because due to the length of the intestine and poor access from the outside, organoid transplants remain limited to the lower colon and have generally 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 enable the introduction of genetic modifications in situ, directly into the normal environment of undisturbed intestinal epithelium, as well as enable the study of the function of multiple genes in normal intestinal epithelium,” stresses Dr. Garside. “This also has advantages because cells in culture can undergo abnormal selection and changes that clutter the determination of gene functions in unmodified cells.”

A new method for studying the intestinal epithelium

Researchers have succeeded in developing a robust and reproducible procedure that makes it possible to introduce a large number of genetic modifications into stem cells of normal, unaltered intestinal epithelium without the need for transplantation. “We used lab mice in our study, which in many respects closely reflect the physiological conditions of the human intestinal epithelium,” says Professor Rudolph.

Due to the difficult-to-reach anatomical location of 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 mice. This is because at this early stage of development, the gut is still turned inside out, making it more accessible for targeted genetic modifications to be introduced.

Huge potential – wide range of applications

The results of the study show that the new technique can be used to study the function of genes in the intestinal epithelium during development and in adult animals. In addition, the method has the potential to identify genes that have an impact on 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 screening is 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 important is that this technique can be applied to other organs derived from the endodermis, such as the pancreas, liver, bladder, and lung, so that the method can be very useful in other areas of research as well. The development of this method is of great interest for the study of these organs and could particularly accelerate discoveries about the biology, function, and progression of diseases of the intestinal epithelium.

Source:

Journal reference:

Garside, GB, et al. (2023) Lentiviral in situ targeting of stem cells in unperturbed intestinal epithelium. BMC Biology. doi.org/10.1186/s12915-022-01466-1.



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