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What cell lines have you injected?
Our lab has injected ES cell lines from a variety of private and commercial labs. These include:
R1 (Nagy) / 129X1/SvJX129S1/Sv+-p)
J1 (129S4/Sv Jae)
D3 (129S2/SvPas)
AB2.2 (129s7/SvEBrd-Hprt b-m2)
RW4 (Incyte/Genome Systems) (129/SvJ)
UCSF Transgenic Laboratory ES cell line
CHZ(129/svEvTac)
MC1 (129s6 SvEv)
How many ES cells do you inject into each embryo?
We inject 12-15 ES cells into each individual embryo. We look for the smallest, roundest, and smoothest cells available to inject. This is our standard protocol. We have found due to variations in cell lines, clones, constructs, and passage number that we will vary the number of cells if we have to do a reinjection. This is because there are instances when the ES cells are not robust enough to overtake the embryo and no chimeric founders are born. The ES cells can also be too robust and will overtake the embryo. Then the embryo does not contribute the proper signaling to carry the fetus to term.
If there are a very low number of chimeras born and a low chimerism rate we will increase the number of ES cells injected into each embryo. We also will increase the number of cells when we start with a clone at a very high passage number, i.e. > 20. If there is a very low pregnancy rate, few pups are born, or the clones are at a low passage number <10-11, we will lower the number of ES cells injected into the embryo.
What strain is the donor embryo?
The host embryo used is a ICR. We collect the embryos at day 3.5 by flushing the uterus. They will soon hatch out of the zona plucida (protein coating membrane) and implant into the uterine horns in the next 24 hours. The embryos are collected, injected, and then cultured for 2-3 hours. We will transfer the embryos into an appropriate pseudopregnant female mouse that same day.
How many embryos do you inject?
We prefer to inject 30-40 embryos per clone. If we inject more than one clone a day we average around 60-70 embryos a day.
What type of mouse do you transfer the embryos into?
We use ICRs as our pseudopregnant mothers. These mice are well known to be excellent mothers, carry large litters, handle surgeries well, and also make excellent foster mothers. These mice are picked in estrus and mated to vasectomized males. They are then pseudopregnant and are receptive to the embryo transfer.
How many embryos do you transfer into each pseudopregnant female?
We transfer 10 embryos into each female. The pups are born 16-17 days later. On average the litters range from 5-8 pups.
Should I breed my female chimeras?
If you targeted a male stem cell line you should have ~80% males and 20% female founders born. The male founders are the ones that you are most interested. A male chimera can be bred to a large number of wild-type females in a short period of time and produce heterozygote mice that much more rapidly. The female chimeras do not always breed, transmit through the germline, and there are published reports that they have genetic problem, i.e. are XXY. If you have a large number of high percentage chimeric males it is probably unnecessary to breed your chimeric females. There is no harm however in mating your chimeric females to wild-type males. There are many cases where female chimeras do produce heterozygotes.
When can I tell if I have chimeras born?
The mice begin to grow fuzzy with hair 5-7 days after birth. When they are fuzzy your can tell which pups are chimeric by coat color. They will appear white and brown with 129 targeted ES cells, and black and white with C57/Bl6 targeted ES cells.
How many chimeras and percentage of chimerism is dependent on a large number of factors. There are clonal variations in each construct. It is not uncommon to get more chimeric founders from one clone to another. This is why we try to inject all your positive clones. Other factors that can have an effect, are what cell line did you target, passage number, and wildtype contamination (which will effect the number of heterozygotes that you produce in germline transmission). These are all factors to consider when you start your gene targeting project. We have been able to produce chimeric founders for of all gene targeted projects brought to our laboratory.