Lentiviral in situ targeting of stem cells in unperturbed intestinal epithelium.
Lentiviral in situ targeting of stem cells in unperturbed intestinal epithelium.
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DOI：
10.1186/s12915-022-01466-1
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发表时间：
2023-01-11
期刊：
影响因子：
5.4
通讯作者：
Rudolph, K. Lenhard
中科院分区：
文献类型：
--
作者：
Garside, George B.;Sandoval, Madeline;Beronja, Slobodan;Rudolph, K. Lenhard
关键词：
在过去的研究中还没有开发出长期原位转导未受干扰的小鼠肠上皮的方法。这种方法可以加速功能研究和筛选，以确定影响肠上皮生物学的遗传因素。在这里，我们开发了一种有效的方法来实现这一长期追求的目标。我们使用超声引导的显微注射，在子宫内第8天（E8.0）的胚胎内胚层。注射过程可在20 min内完成，存活率为100%。通过注射小体积（0.1-0.2 μl）的浓缩病毒，可以成功地转导单个shRNA构建体以及慢病毒文库。新方法稳定且可重复地靶向成人肠上皮，以及其他内胚层衍生的器官，如肺、胰腺、肝脏、胃和膀胱。年轻成年小鼠的出生后分析表明，在E8.0的单个转导细胞在出生后90天产生由每个隐窝区域20-30个相邻隐窝组成的隐窝区域。ApcMin/+突变和野生型小鼠的慢病毒靶向研究表明，Apc功能的杂合缺失抑制了肠隐窝区的发育正常生长模式。这种抑制的隐窝字段的大小并不涉及每个字段的隐窝数量的减少，表明杂合子APC的损失损害了字段内的个别隐窝的增长。慢病毒介导的p53的shRNA敲低导致Apc+/+和ApcMin/+小鼠中每个视野的单个隐窝增加约20%，与ApcMin/+小鼠中隐窝大小的增加有关，但Apc+/+小鼠中隐窝大小略有减少。总的来说，p53基因敲低挽救了APC突变小鼠的隐窝区大小的减少，但对野生型小鼠的隐窝区大小没有影响。这项研究开发了一种新的技术，能够在肠道不同区域的未受干扰的肠上皮中原位靶向肠干细胞。体内体细胞基因编辑和慢病毒库的遗传筛选有可能加快发现和机制理解在发育和出生后生活中控制肠上皮生物学的遗传途径。在线版本包含补充材料，可通过10.1186/s12915-022-01466-1获得。
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. 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 ApcMin/+ 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 ApcMin/+ mice, associating with an increase in crypt size in ApcMin/+ 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. 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. The online version contains supplementary material available at 10.1186/s12915-022-01466-1.
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DOI：
10.1083/jcb.56.1.106
发表时间：
1973-01
期刊：
The Journal of cell biology
影响因子：
--
作者：
Orlic D;Lev R
通讯作者：
Lev R
影响因子：
--
作者：
Nilton A;Sayin VI;Zou ZV;Sayin SI;Bondjers C;Gul N;Agren P;Fogelstrand P;Nilsson O;Bergo MO;Lindahl P
通讯作者：
Lindahl P
影响因子：
46.9
作者：
O'Rourke KP;Loizou E;Livshits G;Schatoff EM;Baslan T;Manchado E;Simon J;Romesser PB;Leach B;Han T;Pauli C;Beltran H;Rubin MA;Dow LE;Lowe SW
通讯作者：
Lowe SW
影响因子：
14.8
作者：
Fumagalli, Arianna;Suijkerbuijk, Saskia J. E.;Drost, Jarno
通讯作者：
Drost, Jarno
影响因子：
2.9
作者：
Kim, Hyeonhui;Kim, Minki;Fang, Sungsoon
通讯作者：
Fang, Sungsoon