DNA: Construction & Purification
PRODUCTION OF TRANSGENIC MICE:
DNA CONSTRUCTION AND PURIFICATION
DNA CONSTRUCT (by Dr. Katya Ravid with members of the core)
OVERVIEW OF CONSIDERATIONS
There are several factors that can influence the success rate of obtaining transgenic animals. The construction of the transgene, the purification of the DNA, the size of the fragment and the concentration of the fragment to be injected all will play a critical role in the expression of your transgene.
There have been occasions where the use of cDNA clones have given expression in transgenic mice, but there are studies that have shown significantly greater success in obtaining expression when your injected construct is genomic DNA rather than cDNA [1&2].
The purification of your construct is absolutely critical for the successful production of transgenic mice. Injected constructs that have degraded will result in a lower percentage of transgenic animals, or they may not work at all. A second isolation and successful purification will often result in a higher number of transgenic animals. Any contaminants from the cloning of the DNA to the isolation of your fragment may be harmful to the embryos and prohibit DNA incorporation and expression. Furthermore, particulate matter in the DNA samples may clog the injection pipettes. There are several procedures that have been successfully used to isolate and purify DNA constructs ; the following are some suggestions with references.
To remove any of the particulate matter that could clog the injection pipettes, all of the solutions that are used to prepare your DNA, isolate and purify the construct should be filtered through a 0.2 µm filter . The vector is purified by CsCl gradient  . Other methods have been shown not to produce the DNA at a consistent purity. The construct is most commonly isolated from the vector using the appropriate restriction enzymes, and gel electrophoresis. This is a result of findings that show the linearization of the gene construct will improve integration , and the removal of extraneous vector sequences will improve the frequency of expression [5&6]. Now your construct can be isolated from the gel by several different methods. Electroelution is still the most commonly used method, but it can be time consuming. There now are several kits available for the isolation of DNA fragments from standard and low melting point agarose gels that have been successful in isolating injection constructs. For constructs 15 kb or larger electroelution is the most recommended method , the glass power method has also been used for larger constructs . For constructs smaller than 15 kb isolation can be carried out using low melt agarose and the Geneclean method (Bio101) , or rapidly becoming very successful is a Qiagen Kit, (QIAGEN, Inc.) for separating DNA fragments from low melt agarose gels. Another alternative method is the Elutip-D column (Schleicher & Schuell Inc.). Once you have isolated your injection construct and diluted it to the correct concentration for injection, a final pass through a 0.2µm syringe filter (Millipore, Cat. No.SLGVL04 OS) will help to remove any remaining impurities that may clog the injection needles.
NOTE: Please contact the facility for additional information if a bacterial DNA will be used.
DNA CONCENTRATION AND AMOUNT
A higher DNA concentration is not necessarily going to increase the percent incorporation of your transgene, however it is important that your DNA is clean. It has been shown that your best percentage of transgene incorporation will occur with a concentration between 1 -5 µg/ml . Most constructs are injected in the range of 2 – 3 µg/ml, and when possible approximately 2 pico-litters are injected into each embryo. Therefore, only 2 -3 µg of DNA are needed for a series of injections, but at least 5µg will be requested to give enough DNA to be run out on a gel to check the quality and purity of your construct. After determining the concentration your linerized injection construct , you can dilute it in injection buffer.
|? Injection Buffer:||10mM Tris, pH 7.4|
|0.1mM EDTA, pH 8.0|
ISOLATING INJECTION CONSTRUCT
DNA Prep for Microinjecting Murine Oocytes
Cut 50 µg of your plasmid to release the fragment, (remove all vector if possible).
Run digest out on an agarose gel, cover the gel box to reduce UV light exposure.
Cut out desired band w/o UV light exposure.
Electroelute in TBE buffer, (slowly as not to heat the buffer past 37 o C).
Concentrate the TBE buffer containing fragment with water saturated butanol to approximately 500 µl.
Spin in micro centrifuge tube for 5 min. @ 4 o C.
Take the top 450 µl and Phenol extract until there is no interface.
Phenol / Chloroform extract twice.
Chloroform extract twice. (You can repeat steps 7-9 to ensure the purity of your fragment).
Bring the fragment to ~ 0.3M NaCl concentration and add twice the volume of 95% ethanol to precipitate the fragment.
Spin at high speed in a micro centrifuge for 5 to 10 min. to pellet the fragment.
Remove as much of the liquid as possible without disturbing your pellet; wash the pellet with 70% ethanol.
Dissolve the pellet in 500 µl of 1M Tris-Cl (pH 7.6) / 0.1mM EDTA (pH 8.0) solution.
Quantitate DNA concentration with UV spectrophotometer and with an agarose gel, injecting fragment should be adjusted to 2-5 µg/ml.
Verify purity of your fragment with a 20 cm gel.
Brinster, R. L., Allan, J. M., Behringer, R. R., Gelinas, R. E. and Palmiter, R. D. (1988) Introns increase transcriptional efficiency in transgenic mice. Proc. Natl. Acad. Sci. USA. 85 : 836 – 840.
Palmiter, R. D., Sandgren, E. P., Avarbock, M. R., Allan, D. D. and Brinster, R. L. (1991) Heterologous introns can enhance expression of transgenic mice. Proc. Natl. Acad. Sci. USA 88 : 478 – 482.
Hogan, B., Costantini, F. and Lacy, E. (1986) Section D. In Manipulating the Mouse Embryo, A Laboratory. Cold Spring Harbor Laboratory. pp. 153 -163.
Brinster, R. L., Chen, H. Y., Trumbuar, M. E., Yagle, M. K. and Palmiter, R. D. (1985) Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc. Natl. Acad. Sci. USA. 82 : 4438 – 4442.
Chada, I. C., Magram, J., Raphael, K.., Radice, G., Lacy, E. and Costantini, E. (1985) Specific expression of a foreign ß – Globin gene in erythriod cells of transgenic mice. Nature. 314 : 377 – 380.
Townes, T. M., Lingrel, J. B., Brinster, R. L. and Palmiter, R. D. (1985) Erythroid specific expression of human ß – Globin genes in transgenic mice. EMBO J. 4 : 17151 – 1723.
Greene, J. R. and Guarente, L. (1987) Subcloning. In : Methods in Enzymology: A Guide to Molecular Cloning Techniques. Abelson, J. N. and Simon, M. L. eds. Academic Press 152 : 512 – 522.
DePamphilis, M. L., Herman, S. K., Martinez-Salas, E., Chalifour, L. E., Wirak, D. O., Cupo, D. Y. and Miranda, M. (1988) Microinjecting DNA into mouse ova to study DNA replication and gene expression and to produce transgenic animals. Biotechniques. 6 : 662 – 680.
Hogan, B., Costantini, F. and Lacy, E. (1986) Section D. In Manipulating the Mouse Embryo, A Laboratory. Cold Spring Harbor Laboratory. pp. 174 -182.
Nagy, A., Gertsenstein, M., Vintersten, K. and Behringer, R., eds. (2003) Manipulating the Mouse Embryo, 3 rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Tymms, M.J. and Kola, I., eds. (2001) Gene Knockout Protocols, Hamana Press, Totowa, New Jersey .
Scientific Director of Transgenic/Knock out Core, Dr. Katya Ravid
Transgenic Specialist and Manager, Greg Martin