University of Washington
Department of Comparative Medicine - HIC - CPP - GNAC - BSL3/ABSL3 - IVS - TRP - UWMCC - UW PRO

Preparation of DNA for Pronuclear Microinjection

Preparation of DNA for Pronuclear Microinjection

General considerations:

The quality of DNA for microinjection is essential to the success of transgenic experiments. DNA that is not purified properly will make the injections difficult and/or reduce the survival. Any traces of phenol, ethanol, salts or enzymes are toxic for embryos. It is also important to get rid of any particles that could clog the injection needles. Glassware with possible detergent residue should be avoided; disposable plasticware rinsed before use with filtered water is used instead.

Prefilter all DNA suspension solutions and microinjection buffer through 0.02 um filters prior to diluting the DNA for microinjection (the 0.02 μm filters will trap DNA molecules) with Anotop syringe filter - Whatman http://www.whatman.com/ 6809-1102. The use of silicone free tubes prevents potential clogging problems.

Sterile endotoxin-free ultra-pure water from Sigma http://www.sigma-aldrich.com/; Gibco http://invitrogen.com or embryo-tested water (e.g. SigmaW1503) should be used for microinjection buffer.

The DNA concentration is a crucial factor to the success of experiments. Excess DNA is toxic and results in developmental arrest. Too diluted DNA will decrease the number of transgenic founders. Errors in concentration can significantly affect transgenic production. The facility requires at least 30ul of DNA fragment at a concentration of at least 50ng/ul. The more concentrated the better, but aim for quality over quantity!

Establish a screening method to identify transgenic animals before submitting DNA for microinjection. To provide evidence of PCR or Southern blot assay that detects your transgene at single copy concentration, dilute transgene DNA into wild-type mouse DNA. A Southern blot assay should be used to determine the copy number, integration site number, and the transgene integrity in the founders.

Procedure:

  1. Purify recombinant plasmid (e.g. Qiagen Endo-Free Plasmid Maxi Kit 12362 or CsCl gradient).
  1. Digest ~50 ug of plasmid with appropriate restriction enzyme, removing as much vector sequences as possible from the insert as they could inhibit the expression of transgene and may be toxic to the zygotes. Run a minigel to check the digest.  Note: If the transgene insert size is similar to the vector size, add enzyme digesting the vector
  1. Separate the insert from the vector on 0.8% agarose gel run in TAE.
  1. Cut out the band of interest using a clean razor blade and transfer the gel slice into a DNase-free Eppendorf tube minimizing DNA exposure to UV light.
  1. Purify the insert by one of the following methods:  

(You may choose to repeat this procedure twice in order to get the cleanest DNA):

  • QIAquick Gel Extraction kit (Qiagen 28704) followed by the QIAquick PCR purification kit (Qiagen 28106)
  • UltraClean™ GelSpin™ Kit (Mo Bio Laboratories 12400-250)
  • Electroelution, ethanol precipitation, ion exchange column (e.g. Elutip-D mini-column Schleicher and Schuell 462615 or Xymotech 27370)
  • GENECLEAN kit (Q.BIOgene 1001-200)
  • NucleoSpinTM Extract Kit (Clontech K3051-1)
  • QIAEX II gel extraction kit (Qiagen 20051)

Use powder-free gloves when handling DNA preparation to avoid potential clogging of injection needles.

  1. Resuspend recovered DNA in filtered microinjection buffer.

*Injection buffer is available from commercial vendors such as CytoSpring

 

Preparation of Microinjection Buffer:

Reagents:

ddH2O, ultrapure, Sigma W1503 (or RNase-free ultra-pure water)

EDTA (0.5M) Sigma E7889

HCL (1M)

Tris-HCL (1M), Sigma T2663

Anotop 10 syringe filter, 0.22um (Whatman 6809-1002)

Method:

  1. Prepare a 5mM Tris, 0.1mM EDTA solution with ultrapure water
  2. Adjust the pH to 7.4 with 1M HCL
  3. Filter buffer through a pre-washed 0.02uM filter
  4. Aliquot and store at 4 degrees or at -20 C

DO NOT use filters that have been sterilized with ethylene oxide!

DO NOT filter nucleic acid solutions through the Anatop filters!

Wash 0.02 um filter with 1 ml buffer – discard wash

Filter remaining buffer through filter for use as dialysis buffer or for use as microinjection buffer.

  1. Determine the concentration and validate DNA fragment
  • Check the concentration and read the 260/280 nm on a NanoDrop. The A260/280 ratio should be 1.8 for pure DNA. Anything lower may indicate the presence of contaminating chemicals such as agarose or ethidium bromide which will be fatal to the injected embryos.
  • Run a minigel with molecular weight markers of known concentration. Make sure that your transgene DNA is intact, of right size and there is no smear of sheared DNA.
  • Adjust the final concentration to the desired 50+ng/ul with FILTERED microinjection buffer. Wash tubes and tips with microinjection buffer or filtered water before use. The final dilution of DNA fragment will be done directly before microinjection by the TRP.

Preparation of BAC/YAC DNA for microinjection:

http://www.med.umich.edu/tamc/BACDNA.html

http://www.cnb.uam.es/~montoliu/prot.html

Saunders, Thomas L. 2009. Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression outcomes. Transgenic Research.

References:

  • Manipulating the Mouse Embryo: A Laboratory Manual. 3rd Ed., by Nagy et al. 2003. Cold Spring Harbor Laboratory Press.
  • Brinster, Ralph and Richard Palmiter. 1985. Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Dev. Biol. 82, 4438-4442.