Development Of A Cre-Loxp Technology With Inducible Uterine Epithelium Specific Activation In Neonatal Mice

In mammals,female uterine glands and their secretions are essential for establishment of uterine receptivity,embryo implantion,and subsequent conceptus survival and growth.Most mammalian female pups are born with only one layer of lumen epithelium but no glands in their utrus.Within a short period of time after birth,some uterine epithelial cells proliferate,differentiate and migrate into the uterine matrix,eventually forming complex tubular uterine glands,known as endometrial gland morphogenesis,or adenogenesis.Many studies have shown that knocking out certain genes result in uterine adenogenesis failure and further lead to infertility.But the underlying mechanisms of glandular development remain largely unknown.e.g.key genes for uterine adenogenesis,and cellular lineage of uterine gland development:whether a cell develops into a gland or many cells co-develop into a gland?To solve these question regarding uterine adenogenesis and gland development,we need an inducible gene engineering tool that can specifically knock-out or knock-in a gene,or mediates cellular lineage tracing at key time points in uterine epithelaium of neonatal mice.We currently have Ltf ^Cre/+gene engineering mouse in our laboratory,withspecific activiation in uterine luminal and glandular epithelium.However,this genetic engineering tool is not an inducible one,and can only mediates gene knock-out and knock-in in uterine epithelium of adult mice but not in neonatal pups.In present study,we want to develop a new technology to allow Ltf ^Cre/+genetic engineering mouse being inducible and able to be used in neonatal mice.This new technology will able to mediates Rosa26-m Tm G or Rosa26-Brainbow reporter mice instantaneously activated or deactivated in neonatal mouse uterine epithelium,which will allow us to trace cellular lineage of uterine glands during adenogenesis by observing expression pattern of fluorescence colors.Two methods were used in this study:inducing of Ltf-Cre activation by subcutaneous injection of estrogen and intrauterine injection of recombinant adenovirus expressing functional Cre enzyme in neonatal mice.For the method of estrogen injection,we first tested effects of estrogen injection on the adenogenesis in wild C57BL/6 mice.Mice were subcutaneously injected with estrogen for three consecutive days from postnatal day 2 to 4（PND2～4）.The results showed that at PND15,the development of uterus and uterine glands in estrogen injected mice was not significantly different from that in control mice.Estrogen doseage ranged from 20 ng,100 ng,200 ng and 400 ng pre mouse were injected subcutaneously into Ltf ^Cre/+Rosa26^{m Tm G/m Tm G}pups from PND2 for three consecutive days.However,early activation of Cre-Loxp system was not observed at PND7 or PND10.This may mainly because of the expression of estrogen receptor alpha in uterine epithelium is relatively low during PND2 to 5,the period when we injected estrogen,which results failure of estrogen induced up-regulation of lactoferri promoter and the activation of Cre enzyme.In the method of intrauterine injection of adenovirus expressing Cre recombinase,a incision was made on the side of Rosa26^{brainbow/brainbow}female pup on PND7,and the uterus was exposed.2μL of adenovirus expressing Cre recombinase with titer of 1*l0¹⁰plaque-forming units per milliliter was injected intrauterinly.The fluorescence expression of uterine epithelium was observed on PND9,11,13,15,17 and 30,respectively.The results showed there is no fluorescence at any time poite.However,strong fluorescence was observed when adenovirus was injected into moue uterine lumen on day 4 of pregnancy,suggesting the uterine epithelial cells of newborn mice may not fit the preconditions for adenovirus infection.In this study,we have tried to developa new inducible Cre-Loxp technology for specific activation in uterine epithelium of neonatal mice,which will provide a powerful tool for the future study of uterine gland development and adenogenesis.By combining this technology with Rosa26^{brainbow/brainbow}genetic engineering mice,we can trace the cellular lineage of uterine endometrial gland in detail way,and further investigate the physiological process of uterine adenogenesis.Although our first attempt of this technology development was unsuccessed,our results can still push the process of this technology one or two steps ahead,and provide research base for further development of this technology.