Transgenic mice produced by Transgenic and Targeted Mutagenesis Laboratory (TTML) are generated by the microinjection of purified DNA into the male pronucleus of single-cell fertilized mouse embryos (zygotes). Microinjected embryos are then surgically transferred into the reproductive tract of a pseudopregnant female who carries the pregnancy to term.
Transgenic DNA integrates randomly into the embryonic genome following microinjection. Usually, multiple copies of the transgene integrate at a single chromosomal site just prior to the first cell division, which ensures that the transgene is present in all nucleated cells of the developing embryo. Approximately 10 to 30 percent of the microinjected embryos that develop to term will inherit the transgene. These founder (F0) mice, although carrying the same transgene, are unique as they vary from each other in the number of transgenes that integrate (transgene copy number) and the chromosomal site at which the transgenes integrated (integration site). Both the integration site and, to a lesser extent, the transgene copy number can affect transgene expression patterns, which must be determined for each newly created line.
Follow the links below or review our Frequently Asked Questions section for more information.
Frequently Asked Questions
Will all the pups born following microinjection carry the transgene?
No, integration of the microinjected transgenic DNA into the embryonic genome is not very efficient. Therefore, only about 10 to 30 percent of pups will have inherited the transgene. Since integration most often occurs prior to the first zygotic cell division, the transgene will be present in all nucleated cells of those mice, the founder (F0) mice, that inherit the transgene. While all of the founder mice carry the exact same transgene, integration of the transgene is a random event. Therefore, each founder is considered unique with respect to the chromosomal location or integration site of the transgene, the number of transgene copies integrated at that site and the expression pattern of the transgene.
Do all the founders that inherited the transgene express the transgene?
No, the chromosomal environment at the transgene integration site can significantly influence transgene expression, a phenomenon called position effect variegation. For example, if the transgenic DNA integrates into heterochromatin, which is transcriptionally inactive, the transgene will not be expressed in the adult mouse. As integration is a random event, transgenic expression levels and patterns can vary substantially among founders.
Sequences within the transgene can also effect expression. Vector sequences of 100bp or more may be recognized as foreign and target the locus for transcriptional inactivation, whereas the inclusion of heterologous introns or insulator sequences can enhance expression.
I have bred my founder several times and none of the progeny inherit the transgene. Why is this?
Occasionally, the DNA integrates after the first cellular division resulting in some tissues/cells that carry the gene and others that do not. These mosaic founder animals may transmit the transgene through the germline at very low frequencies.
Now that I have my founders, what should I do?
Remember that each founder, although carrying the same transgene, is unique with respect to copy number and integration site. For these reasons, unless there is a specific purpose, breeding one founder to another is not recommended.
The relative levels and tissue-specific pattern of transgene expression must be established for each founder line. Expression patterns are commonly analyzed in F1 mice as transgene expression products, such as mRNA or protein, and often require the isolation of tissue. This information will help to determine which founder lines should continue to be bred for proposed experiments. For instance, tissue-specific expression may not be tightly regulated in some founder lines resulting in non-specific or mis-expression of the transgene in various tissues. Expression levels may be too high or too low in other founder lines making these lines unsuitable for further analysis.
What strain should I use to generate F1 mice for expression analysis?
Founder mice can, in general, be bred to any desired strain. We recommend mating wild-type mice, not non-transgenic littermates to founders initially to begin establishing the line and generating F1 for expression analysis. Mixed B6:SJL founders can be kept on a B6SJLF1 background or crossed with C57BL/6 mice if interested in backcrossing the transgene onto a pure background strain.