Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

The overall goal of the following experiment is to dissect gene function in different stages of terminal erythropoiesis using an in vitro mouse fetal liver culture system. This is achieved by first purifying TER one 19 negative mouse fetal liver erythroblasts from embryonic day 13.5 pregnant mice As a second step, the purified erythroblasts are transduced with virus encoding CD NA or S-H-R-N-A of the gene of interest to study the gain or loss of function of the gene respectively. Finally, the transduced TER one 19 negative cells are cultured under one of two different conditions to dissect the function of the gene in the early and late stages of terminal erythropoiesis.

Ultimately, the different kinetics of the differentiation and the enucleation of the transduced cells can be evaluated by flow cytometric analysis. So this method can provide insight into mouse atrial osis. It can also be applied to other HESIs hepo systems such as lympho, osis, and myosis.

To purify the fetal liver erythroblasts begin by disinfecting the abdomen of a euthanized E 13 to E 15 timed pregnant mouse with 70%ethanol. Then open the abdomen with dissecting scissors to remove the uterus and place the uterus in PBS. Next in a tissue culture hood, using sterile tools, dissect out the fetuses and place them in PBS.

Then holding the body of one fetus at a time with one forceps. Gently pull each pink fetal liver away from the body with another forceps. Use the forceps to clean the associated fibrotic tissues from each liver and then place the livers into fresh PBS containing 10%FBS mechanically disrupt the livers by pipetting the tissue up and down a few times, and then filter the resulting cell suspension through a 40 micrometer cell strainer.

After spinning down the cells for five minutes at 800 Gs at four degrees Celsius, resuspend the pellet in one milliliter of PBS and FBS. To purify the TER one 19 negative erythroblasts, block the cells with rat IgG on ice. After 15 minutes, add biotinylated anti TER one 19 antibody to the cell suspension for another 15 minutes.

Then after washing the cells in PBS and FBS Reese, suspend the cells in one milliliter of fresh PBS plus FBS and incubate the cells in 75 microliters of streptavidin conjugated with magnetic particles on ice. After 10 minutes, bring the total volume up to 2.5 milliliters with PBS plus FBS, and then transfer the cell suspension into a five milliliter polypropylene round bottom tube. Insert the tube into a magnetic cell sorting apparatus, and then after another 10 minute incubation, pour the unattached TER one 19 negative erythroblasts into a new five milliliter tube after another incubation.

To remove the residual TER one 19 positive cells, spin down the TER one 19 positive erythroblasts. Resuspend the cells in one milliliter of PBS containing FBS and count the live cells with trian blue to transduce the purified TER one 19 negative fetal liver cells first plate them at three to 500, 000 per well in a fibrin actin coated 12 well plate then add poly brain to a final concentration of 10 grams per milliliter to facilitate the viral transduction and spin fect the cells with the virus of interest for one to 1.5 hours at 800 GS and 37 degrees Celsius. To evaluate gene function in the early stage of terminal erythropoiesis cultured the transduced cells in EPO free stem cell factor medium for 12 hours.

Then after spinning down the cells, Reese spend the cells in each well with one milliliter of EPO containing medium and culture them for 24 to 48 hours. The cells can then be harvested for further analysis to test gene function in the late stage of terminal erythropoiesis culture. The transduced cells immediately in EPO containing medium and harvest the cells after 24 to 48 hours of culture for further assays in these density plots, the purity of the fetal liver erythroid cells before and after magnetic bead sorting is shown for the early stage of terminal erythropoiesis.

After viral transduction, the cells are cultured in EPO free stem cell factor medium for 12 hours. During this period, the cells maintain their progenitor status. The transduced genes were expressed after 12 hours.

In SCF medium, no significant apoptosis was observed in the GFP transduced cells during the two day culture in EPO containing medium in both methods. Most of the CD 71 low TER one 19 low erythroid progenitor cells exhibit induction of the transferrin receptor and TER one 19. By day two, enucleation occurs on day two as observed on the hosts high TER one 19 high erythroblasts and hosts low TER one 19 high retic lytes.

It is notable that cells immediately cultured in EPO more rapidly differentiate and nucleate than cells cultured first in stem cell factor medium Following this procedure. Other method like cell proliferation, assays or western blood analysis can be performed to determine the cellular or molecular responses after oral expression or regulations of genes of interest.