Lentiviral in situ targeting of stem cells in unperturbed intestinal epithelium
Abstract Background Methods for the long-term in situ transduction of the unperturbed murine intestinal epithelium have not been developed in past research. Such a method could speed up functional studies and screens to identify genetic factors influencing intestinal epithelium biology. Here, we dev...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-01-01
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| Series: | BMC Biology |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12915-022-01466-1 |
| _version_ | 1797952332248383488 |
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| author | George B. Garside Madeline Sandoval Slobodan Beronja K. Lenhard Rudolph |
| author_facet | George B. Garside Madeline Sandoval Slobodan Beronja K. Lenhard Rudolph |
| author_sort | George B. Garside |
| collection | DOAJ |
| description | Abstract Background Methods for the long-term in situ transduction of the unperturbed murine intestinal epithelium have not been developed in past research. Such a method could speed up functional studies and screens to identify genetic factors influencing intestinal epithelium biology. Here, we developed an efficient method achieving this long-sought goal. Results We used ultrasound-guided microinjections to transduce the embryonic endoderm at day 8 (E8.0) in utero. The injection procedure can be completed in 20 min and had a 100% survival rate. By injecting a small volume (0.1–0.2 μl) of concentrated virus, single shRNA constructs as well as lentiviral libraries can successfully be transduced. The new method stably and reproducibly targets adult intestinal epithelium, as well as other endoderm-derived organs such as the lungs, pancreas, liver, stomach, and bladder. Postnatal analysis of young adult mice indicates that single transduced cells at E8.0 gave rise to crypt fields that were comprised of 20–30 neighbouring crypts per crypt-field at 90 days after birth. Lentiviral targeting of Apc Min/+ mutant and wildtype mice revealed that heterozygous loss of Apc function suppresses the developmental normal growth pattern of intestinal crypt fields. This suppression of crypt field sizes did not involve a reduction of the crypt number per field, indicating that heterozygous Apc loss impaired the growth of individual crypts within the fields. Lentiviral-mediated shRNA knockdown of p53 led to an approximately 20% increase of individual crypts per field in both Apc +/+ and Apc Min/+ mice, associating with an increase in crypt size in Apc Min/+ mice but a slight reduction in crypt size in Apc +/+ mice. Overall, p53 knockdown rescued the reduction in crypt field size in Apc-mutant mice but had no effect on crypt field size in wildtype mice. Conclusions This study develops a novel technique enabling robust and reproducible in vivo targeting of intestinal stem cells in situ in the unperturbed intestinal epithelium across different regions of the intestine. In vivo somatic gene editing and genetic screening of lentiviral libraries has the potential to speed up discoveries and mechanistic understanding of genetic pathways controlling the biology of the intestinal epithelium during development and postnatal life. The here developed method enables such approaches. |
| first_indexed | 2024-04-10T22:44:40Z |
| format | Article |
| id | doaj.art-33b2e89d547c4e1a91b000ef317c7a21 |
| institution | Directory Open Access Journal |
| issn | 1741-7007 |
| language | English |
| last_indexed | 2024-04-10T22:44:40Z |
| publishDate | 2023-01-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biology |
| spelling | doaj.art-33b2e89d547c4e1a91b000ef317c7a212023-01-15T12:21:32ZengBMCBMC Biology1741-70072023-01-0121111210.1186/s12915-022-01466-1Lentiviral in situ targeting of stem cells in unperturbed intestinal epitheliumGeorge B. Garside0Madeline Sandoval1Slobodan Beronja2K. Lenhard Rudolph3Leibniz Institute on Aging, Fritz Lipmann InstituteDivision of Human Biology, Fred Hutchinson Cancer CenterDivision of Human Biology, Fred Hutchinson Cancer CenterLeibniz Institute on Aging, Fritz Lipmann InstituteAbstract Background Methods for the long-term in situ transduction of the unperturbed murine intestinal epithelium have not been developed in past research. Such a method could speed up functional studies and screens to identify genetic factors influencing intestinal epithelium biology. Here, we developed an efficient method achieving this long-sought goal. Results We used ultrasound-guided microinjections to transduce the embryonic endoderm at day 8 (E8.0) in utero. The injection procedure can be completed in 20 min and had a 100% survival rate. By injecting a small volume (0.1–0.2 μl) of concentrated virus, single shRNA constructs as well as lentiviral libraries can successfully be transduced. The new method stably and reproducibly targets adult intestinal epithelium, as well as other endoderm-derived organs such as the lungs, pancreas, liver, stomach, and bladder. Postnatal analysis of young adult mice indicates that single transduced cells at E8.0 gave rise to crypt fields that were comprised of 20–30 neighbouring crypts per crypt-field at 90 days after birth. Lentiviral targeting of Apc Min/+ mutant and wildtype mice revealed that heterozygous loss of Apc function suppresses the developmental normal growth pattern of intestinal crypt fields. This suppression of crypt field sizes did not involve a reduction of the crypt number per field, indicating that heterozygous Apc loss impaired the growth of individual crypts within the fields. Lentiviral-mediated shRNA knockdown of p53 led to an approximately 20% increase of individual crypts per field in both Apc +/+ and Apc Min/+ mice, associating with an increase in crypt size in Apc Min/+ mice but a slight reduction in crypt size in Apc +/+ mice. Overall, p53 knockdown rescued the reduction in crypt field size in Apc-mutant mice but had no effect on crypt field size in wildtype mice. Conclusions This study develops a novel technique enabling robust and reproducible in vivo targeting of intestinal stem cells in situ in the unperturbed intestinal epithelium across different regions of the intestine. In vivo somatic gene editing and genetic screening of lentiviral libraries has the potential to speed up discoveries and mechanistic understanding of genetic pathways controlling the biology of the intestinal epithelium during development and postnatal life. The here developed method enables such approaches.https://doi.org/10.1186/s12915-022-01466-1Genetic engineeringMicroinjectionIntestineAPCTransductionIntestinal stem cells |
| spellingShingle | George B. Garside Madeline Sandoval Slobodan Beronja K. Lenhard Rudolph Lentiviral in situ targeting of stem cells in unperturbed intestinal epithelium BMC Biology Genetic engineering Microinjection Intestine APC Transduction Intestinal stem cells |
| title | Lentiviral in situ targeting of stem cells in unperturbed intestinal epithelium |
| title_full | Lentiviral in situ targeting of stem cells in unperturbed intestinal epithelium |
| title_fullStr | Lentiviral in situ targeting of stem cells in unperturbed intestinal epithelium |
| title_full_unstemmed | Lentiviral in situ targeting of stem cells in unperturbed intestinal epithelium |
| title_short | Lentiviral in situ targeting of stem cells in unperturbed intestinal epithelium |
| title_sort | lentiviral in situ targeting of stem cells in unperturbed intestinal epithelium |
| topic | Genetic engineering Microinjection Intestine APC Transduction Intestinal stem cells |
| url | https://doi.org/10.1186/s12915-022-01466-1 |
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