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Short review of cytoplasmic injection method, plus injection pipette bevelling hint

"Transgenesis in mice by cytoplasmic injection of polylysine/DNA mixtures"; Transgenic Research, Vol. 4, pp353-360 (1995); Raymond L. Page, Stephen P. Butler, Anuradha Subramanian, Francis C. Gwazdauskas, John L. Johnson and William H. Velander.

This paper offers an alternative to pronuclear microinjection in order to produce transgenic animals, by using a methodology which is of particular use in animal species where visualization of the pronuclei is difficult.

There are many inherent problems with the transfer of DNA into whole mammalian organisms, either by embryonic stem cells (labor intensive, high frequency of germ line mosaicism), retroviral infection (small DNA "payload") or pronuclear injection (genome replication prior to injection leading to somatic and germline mosaicism). Although pronuclear injection has been studied extensively, the mechanism by which integration occurs is poorly understood, although it is suggested that it may be a random process dependent on a breakage-repair pathway. Injection of DNA into the cytoplasm has no effect on transgenesis, suggesting that the mechanism allowing integration to occur is located within the pronuclei (for attempts in this area, see Brinster et al, 1985; PNAS Vol. 82, pp4438-42). Other attempts to generate transgenic animals by cytoplasmic injection (liposome encapsulation by adsorption / microinjection) have been unsuccessful.

However, using the established method of DNA-ligand-polylysine conjugate to transfect mammalian cells / DNA-transferrin-polylysine transfecting avain cells it was suggested that this might provide a means for cytoplasmic injection to be successful. This paper reports the success of cytoplasmic injection of DNA/polylysine mixtures in producing transgenic animals, and also examines the effects of a number of DNA concentrations and lysine to phosphate ratios on transgenic efficiency.

Methods

Zygote manipulation was described as superovulating CD-1 mice (at 24-30 days old), collecting the embryos by standard methodology. Embryos were washed in M2 media and retained in a paraffin oil-covered drop of media, visualized on an inverted microscope at 200X where DNA was injected into the cytoplasm by standard methodology. Injected embryos were then either cultured or implanted into pseudopregnant mice. DNA preparation was carried out by cloning the injection construct in pUC-19, restricted and purified (HPLC), precipitated in microinjection buffer and stored at 2X the concentration to be used for the injection experiments. Polylysine (bromide salt, 51-mer) was dissolved in injection buffer and DNA/polylysine mixtures yielding lysine to phosphate ratios (L:P) of 0.5, 1.0 and 2.0 were made, mixing equal volumes of polylysine and DNA. The mixtures were allowed to stand for at least 15 minutes at room temp. prior to loading into injection needles.

Production

Approximately 10pl (using pneumatic microinjector - Eppendorf 5242) was injected into the cytoplasm of each zygote, with care to avoid any pronuclei. DNA from the resultant pups born from this study was analysed by PCR for the presence of the transgene. PCR positive mice were confirmed by DNA in-gel hybridization. Initial founders were evaluated for germ line transmission.

Results

The L:P ratios were tested by freezing embryos immediately after injection of differing DNA/polylysine mixtures (with different DNA concentrations also), and performing PCR on them. At L:P ratios of 0.5:1 and 1:1 the DNA construct was detected in 100% and 50% respectively, with only 2 of 30 zygotes at L:P of 2:1 showing evidence of the presence of the injected construct.

The transgenesis efficiency can be summarized in the following table:- (view thru HTML browser)

DNA concentration (ug ml -1)
Injection Site L:P 1.5 15 50
Pronucleus0:15/23 (21.7%)ab
Cytoplasm0:10/8 (0.0%)0/15 (0.0%)0/53 (0.0%)
Cytoplasm0.5:12/56 (3.6%)0/54 (0.0%)4/37 (10.8%%)
Cytoplasm1:10/35 (0.0%)2/23 (8.7%)6/47 (12.8%)
Cytoplasm2:10/25 (0.0%)4/58 (6.9%)2/39 (5.1%)
 Effect of DNA concentration and polylysine on integration frequency for mice born from microinjection into the cytoplasm

a,b = above 5 ug ml -1 embryo/pup viability is reduced significantly (Brinster et al, 1985)

Injection Site
Parameter Pronuclear, DNA alone Cytoplasm, polylysine/DNA mixture Cytoplasm, DNA alone
Zygotes injected262451293
Zygotes transferred143246205
Pups born234745
Transgenic pups560
Transgenic efficiency*21.7%12.8%0.0%
Overall efficiency**3.6%2.4%0.0%
 Comparison of the efficiency of producing transgenic mice by the cytoplasmic injection of either DNA alone (50 ug ml -1) or a DNA/polylysine solution (50ug ml -1, 1:1 L:P ratio) to pronuclear microinjection of DNA (1.5 ug ml -1)

* = Transgenic efficiency is the % of pups born testing positive for the transgene.

** = Overall efficiency is the % of zygotes transferred that result in pups testing positive for the transgene.

The take-home message for this paper is that cytoplasmic injection of DNA is possible by the method described, although there is a loss of efficiency over the standard pronuclear injection procedure. The major caveat is that injection of zygotes is possible when the pronuclei are not visible, be this as a result of early or late embryo isolation, or using a species where pronuclear visualization is problematical (sheep, pigs, cattle).

There is also the unexplored avenue that injection of DNA/polylysine mixtures anywhere in the zygote (including the pronucleus) may yield transgenic animals, thereby allowing trainee injectors a chance to be productive while acquiring the skills necessary to complete pronuclear injection proper.

Helpful hint


This helpful hint is the result of an article in Biotechniques, Vol.14, No.3, pp412-414. This was first introduced to the transgenic community in St. Louis at Washington University during my own stint there in the mid-90s. This article is a report by Kristian Gundersen, Theresa Hanley and John Merlie, on the use of micropipette bevelling to increase transgenic (pronuclear injection) embryo yield. A slurry of grit 120 silicon carbide is used in a beaker on a stir plate to provide a gentle yet abrasive force. Micropipettes drawn out previously on a vertical or horizontal puller can be further modified by the immersion of their tips into the rotating slurry at approximately 45 degrees for 15-30 seconds. This imparts an aperture which is less prone to clogging and also reduces the shearing force applies to the DNA as it is expelled through the glass needle. These factors together serve to increase the viability of injected eggs and the subsequent successful transgene integration. See Techniques and Protocols "Micropipette Bevelling"


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