| First Author | Lo CW | Year | 1992 |
| Journal | Mouse Genome | Volume | 90 |
| Pages | 684-686 | Mgi Jnum | J:123337 |
| Mgi Id | MGI:3718091 | Citation | Lo CW, et al. (1992) Iontophoretic DNA injections and the production of transgenic mice. Mouse Genome 90:684-686 |
| abstractText | Full text of Mouse Genome contribution: Iontophoretic DNA injections and the Production of Transgenic Mice. Cecilia W. Lo, Roberto Diaz, and Colleen Kirby. Biology Department, University of Pennsylvania, Philadelphia, PA 19104-6017. Transgenic mice usually are made using pressure injection methods to microinject DNA into the pronuclei of mouse eggs (1). Iontophoretic injection can be used as an alternative method for microinjecting DNA into mouse eggs (2). With microinjection mediated by iontophoresis, the DNA is expelled into the pronucleus with a small current pulse. As there is little net fluid displacement, this method of microinjection may be advantageous in minimizing physical shearing of the DNA molecule. To examine the feasibility of using this injection method to generate stable transgenic mouse lines, we iontophoretically injected the plasmid pMBJ into the pronuclei of mouse eggs (C57BL6/J X SJL/J). This pBR322 based plasmid contains a 7.0 kb mouse genomic Eco RI fragment which includes the mouse Beta-major globin gene (3). Injections were carried out using microelectrodes with tip sizes of 0.1 um, and the DNA was ejected using 5-10 nA current pulses of 0.5-2.0 min duration. 80% of the injected eggs were viable after injection, and of these 30% produced live births after transfer into foster mothers. Southern analysis of tail DNA from 85 mice revealed that three contained pMBJ derived DNA inserts. These three founder transgenic mice are referred to as animals 43, 59, and 83 (Fig. 1). The DNA inserts in these mice are stably heritable through the germ line, and all three founders are mosaic for the exogenous DNA inserts (compare band intensity of founder vs. offspring samples in Fig. l ; 4). In two animals, 43 and 83, most of the DNA inserts are organized as a long tandem array. Thus Eco RI digestion of genomic DNA from 43 and 83 revealed two very prominent pMBJ hybridizing fragments - a 4.4 kb band which corresponds to the pBR vector DNA (and hybridizes exclusively to pBR), and a 7.0 kb fragment (not hybridizing with pBR) corresponding to the mouse genomic insert of pMBJ (Fig. 1 and data not shown). The identity of these exogenous DNA inserts as being pMBJ derived and their tandem arrangement was confirmed with additional restriction digestion and Southern blot analysis (data not shown). Densitometric analysis of Southern blots show that in the offspring of line 83, the tandem array contained greater than 800 copies of the pMBJ plasmid, while for line 43, greater than 150 copies of the pMBJ plasmid DNA was incorporated in the tandem repeat. In line 59 which exhibited no evidence of tandem inserts, the exogenous DNA segregated as two separate linkage groups (59a and 59b) (Fig. 1a), thereby indicating that the founder animals contained two independent chromosomal insertions. The chromosomal location of the exogenous inserts in each of the transgenic lines were characterized by in situ hybridization of metaphase chromosome spreads obtained from the bone marrow. For these experiments, a tritiated nick translated pMBJ derived probe, pMBd2, was used. (4). With 83 metaphase spreads, strong hybridization signal was observed at the telomeric end of chromosome 3 (see Fig. 2c, d), while with 43 metaphase spreads, strong hybridization signal was localized to the pericentromeric region of chromosome 17 (see Fig. 2a, b). Similar analysis of metaphase spreads from offspring 59a and 59b revealed that for 59b, the exogenous inserts was localized to the distal third of chromosome 3, and for 59a, the pericentromeric region of chromosome 5 (data not shown). Further analysis of blood samples from these mice showed that the Beta major globin chains are not expressed in the hemoglobins found in these animals (data not shown). This is consistent with the previous reports that pBR322 DNA can be inhibitory to the expression of transgenes in mice (5, 6). Figure 1. (Legend). Southern blot of tail DNA f'rom transgenic founders and offspring. (a, b) Eco RI digests of DNA from transgenic founder 43, 59, 83 and their respective offspring 43/a, 59/a, 59/b, and 83/a were analyzed by Southen blot hybridization with a pMBJ derived probe. As expected, each sample exhibited the endogenous Beta-globin band at 10.5 kb. In addition, several other exogenously derived bands were detected, including very prominent 4.4 and 7.0 kb bands in transgenic line 43 and 83. (a) represents a shorter exposure of the blot shown in (a). Figure 2. (Legend). Metaphase chromosome in situ hybridization. (a, c) Banded metaphase spreads from transgenic line 43 and 83 prior to hybridization. (b, d) Same metaphase spreads after hybridization with a pMBJ derived hybridization probe. The very high copy tandem insertion found in transgenic line 83 appears to be the largest reported for any transgenic mouse line (at least to our knowledge). It should be noted that in conjunction with the tandem array, there are also many other pMBJ derived fragments present in the exogenous DNA insert of this transgenic line (see Fig. 1b). This very large amount of exogenous DNA in line 83 does not appear to be deleterious to the animal, as 83 has been bred and maintained in the homozygous state for many generations. Moreover, we have observed no overt phenotype in line 83. As the high copy insert found in line 83 is readily detectable by in situ hybridization, not only in metaphase spreads but also in tissue sections (7), the DNA marker in this transgenic line has been successfully used for tracking cell lineages (4, 8, 9), with no evidence of non-cell autonomy or lineage bias noted (8, 9). In future studies, it will be interesting to examine whether other transgenic lines containing high copy inserts similar to those in line 83 can be generated by iontophoretic injection. Such marked chromosomes may be useful not only for lineage studies but possibly also other analyses where tracking of cells or chromosomes may be necessary. It also will be interesting to examine whether iontophoretic DNA injection may facilitate the production of transgenic mice containing large DNA molecules, such as those derived from yeast artificial chromosomal (YAC) vectors. With iontophoresis, it is possible that some of the difficulties associated with the microinjectron of YACs can be overcomed. Thus with electrical current used to displace DNA from the micropipette, physical shearing of the DNA may be minimized and the high back pressure experienced at the injection tip may be alleviated. We are currently carrying out additional studies to examine these possibilities. Acknowledgements This work was supported by N.T.H. grants GM30461 and HD21355. References 1. Palmiter, R.D., and Brinster, R.L. 1986. Annu. Rev. Genetics 20:465-500. 2. Lo, C.W. 1983. Mol. Cell Biol. 3:1803-1814. 3. Tilghman, S.M., Tiemeier, D.C., Polsky, F., Edgell, M.H., Seidman J.G., Leder, A., Enquist, L.W., Norman, B., and Leder, P. 1977. Proc. Natl. Acad. Sci. 74:4406-4410. 4. Lo, C.W. 1987. Differen. 35:37-44. 5. Chada, K., Magram, J., Raphael, K., Radice, G., Lacey #., and Costantini, F. 1985. Nature 314:377-380. 6. Hammer R.E., Krumlauf, R., Camper, S.A., Brinster, R.L., and Tilghman, S.M. 1987. Science 235:53-58. 7. Lo, C.W. 1986. J. Cell Sci. 818:143-162. 8. Thomson, J.A., and Solter, D. 1988. Roux's Arch. Dev. Biol. 197:63-65. 9. Clarke, H.J., Varmuza, S., Prideaux, and Rossant, J. 1988. Develop. 104:175-182. |
