The generation of transgenic animals by DNA microinjection is a powerful tool to investigate the molecular regulation of gene expression, development, and disease.

The power of this technology is that foreign DNA can be introduced into every cell of a developing organism including germ line and can be transmitted to the offspring.

 We offer:

Pronuclear microinjection of DNA is the most common and extensively used animal transgenesis method. In this technique, the engineered transgene is injected directly into the pronuclei of fertilized zygotes. In order to obtain fertilized zygotes, superovulation in female mice/rats is induced with gonadotropins. After mating with males, embryos are harvested from females with present vaginal plug. Subsequently, embryos with two visible pronuclei are injected with the DNA construct of interest. Zygotes that survive the procedure are transferred into pseudopregnant females. After 3 week pregnancy, potentially transgenic animals are born. Tissue samples for genotyping are collected from 3 week-old pups.

 

EXPERIMENT FLOWCHART

 

  On a routine procedure we use C57Bl6 or FVB/N mice and Wistar rats strains as embryo donors. Nevertheless, any other strain can be used but this may require additional time and costs.

 

DNA microinjection into pronuclei usually leads to generation of founders with randomly integrated concatamers of transgenes. Thus, every single founder should be treated as a separate line, as the number of copies and integration site will vary between them. This will probably have an effect on different levels of transgene expression and inheritance frequency. Described disadvantages can be omitted by using targeted transgenesis method, designed to insert single-copy transgenes into specified loci. Currently, we are introducing this technique into our Laboratory services.

Lentiviral injection - subzonal injection

Lentiviral vectors can be produced in LAM Transgenic Facility and injected through zona pellucida into perivitelline space without harming the embryo. Viral vectors use their natural mechanisms for penetration into the cell and incorporation  to the host genome. Embryos injected in this method are subsequently transfer to the foster mothers and genotype after wean from mother.

 

Sample preparation

Genetically engineered transgene of interest should be verified before being used for microinjection. We recommend to sequence and express the construct in vitro. This will not guarantee its expression in vivo, however, it will enable detection of possible errors, mutations, etc.

 Proper purification of transgenic expression cassettes is crucial for successful transgenic animal production. Embryos are highly susceptible to trace contaminants such as phenol, ethanol, endotoxin, ethidium bromide, agarose and enzymes. Poor purification of DNA may cause technical problems with microinjection such as obstruction injection needle and decrease survival rate of embryos after procedure.

 LAM offers plasmid preparation for microinjection but when investigator decides to prepare DNA, we recommend some commercial available kits:

http://products.invitrogen.com/ivgn/product/K200025

https://www.qiagen.com/no/shop/sample-technologies/dna/qiaex-ii-gel-extraction-kit/#orderinginformation

Protocol for plasmid DNA purification:

Perform restriction enzyme digestion of 50μg of a cloning vector. Run a few nanograms of digest mixture on a gel to determine reaction efficacy.  After successful digestion with restriction enzymes and verifying the bands’ molecular weight are as you expected, you should run the electrophoresis, excise from the gel the band of interest and purify it. DNA should be eluted with filtered TE Embryo (10mM Tris-HCl, pH 8.0, 1mM EDTA) and the concentration should  be checked (prepare the sample as concentrated as the kit allows).  Do not dilute the sample. Quantify eluted DNA by measuring A260.  Determine the A260/ A280 ratio. A ratio of ≥1.8 is perfect for injections.

Protocols for BAC DNA purification:

Methods for BAC preparation are available in the link attached below:

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

You may submit enzymatically liberated and purified transgene, ready for microinjection.

A photo gel with marked and described bands should be submitted together with the sample.

When submitting a sample, a photo gel of your genotyping PCR should be attached.
All documents (MI submission form, gel photos, etc.) can be mailed together with the sample, brought directly to our laboratory, or scanned and e-mailed in advance

Microinjection submission form: MI_order_form.pdf

Turnaround times
After obtaining DNA ready for microinjection, the minimum time required for generating transgenic founders is around 8-12 weeks. This, however, can vary depending on the current work schedule, as well as on the failure in the production of founders. If the latter occurs, we decide about further steps together with investigator.

Contact us for further details -

a.klejman@nencki.gov.pl

j.chilczuk@nencki.gov.pl