Design and Preparation of DNA for Microinjection

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3.3.1. Design of DNA Construct

Most transgene constructs contain cDNAs of the protein to be overexpressed under the control of a promoter suitable for the intended tissue distribution of the transgene product. A schematic drawing of a typical transgene construct is shown in Fig. 1. Table 1 summarizes promoters already used successfully in transgenic experiments to express genes in specific cell types important for cardiovascular regulation.

In order to increase the expression of the transgene (19), an intron should be included in the construct, if the promoter does not already contain it such as some cardiac specific promoters do. Most commonly used for this purpose are the small intron of simian virus 40 (SV40) and the rabbit P-globin intron. In our experience, placement of the intron between promoter and cDNA is preferable to its insertion behind the cDNA. In our hands the use of the P-globin

Table 1

Promoters Commonly Used to Target Transgene Expression to Specific Cell Types Important for Hypertension Research

Promoter Abbreviation Cell type of highest activity Reference

Table 1

Promoters Commonly Used to Target Transgene Expression to Specific Cell Types Important for Hypertension Research

Promoter Abbreviation Cell type of highest activity Reference

Myosin light chain 2

MLC-2

Cardiomyocytes

28

a-Cardiac myosin

heavy chain

aMHC

Cardiomyocytes

29

tie2

tie2

Endothelial cells

30

SM22a

SM22a

Vascular smooth muscle cells

31

Smooth muscle a-actin

SMA

Smooth muscle cells

32

Smooth muscle

SM-MHC

Smooth muscle cells

33

myosin heavy

chain

Kidney androgen-

KAP

Renal proximal tubular cells

34

responsive

protein

Sodium chloride

TSC

Renal distal tubular cells

35

cotransporter

Tamm Horsfall

THP

Renal tubular cells

36

protein

Neuron-specific

NSE

Neurons

37

enolase

Tubulin a-1

T-a1

Neurons

38

Ca2+/calmodulin-

CaMKII

Neurons

39

dependent

kinase II

Glial fibrillary

GFAP

Astrocytes

40

acidic protein

intron or of a promoter including introns generally resulted in better transgene expression than the use of the SV40 intron (see Note 8). To allow for correct transcriptional termination a polyadenylation [poly(A)] site should be included at the 3' end of the construct. Here, poly(A) sites of SV40 or bovine growth hormone are among the most frequently used. Note, that the construct must be released from vector sequences when completed; thus there should still be suitable restrictions sites left for this purpose (see Note 9).

3.3.2. Choice of Microinjection Buffer

A very important factor determining embryo survival is not only the injected DNA itself but the solution buffer of the DNA. There are many studies dealing with the optimal solution for dissolving DNA for microinjection. Researchers have even used water or 1X phosphate-buffered saline (PBS) for this purpose (20,21). However, in most laboratories, including ours, Tris-EDTA buffer is used.

3.3.3. Purification of DNA-Construct for Microinjection

Success of microinjection depends on the method used for DNA preparation (22). Vector-free DNA is isolated by restriction enzyme digestion followed by agar gel electrophoresis. Isolation of DNA from the agar gel by binding to glass beads followed by passage through a Sephadex G-50 column (QIAquick kit from Qiagen) and filtration through a 0.22-^m filter provides DNA of high quality.

The concentration of the DNA to be injected has been found to be a major criterion for embryo survival and efficiency of transgene integration (6). A DNA concentration of 1 ng/^L was less detrimental to embryo survival than 10 ng/^L and lead to a higher rate of transgene integration than 0.1 ng/^L. A concentration of 3-5 ng/^L is most commonly used for the production of transgenic mice and rats.

1. Digest approx 20 ^g of the DNA construct with restriction enzymes that will remove the plasmid sequences from the transgene.

2. Run the DNA on an agarose gel and elute the band containing the transgene to be injected. We have used a Qiaquick gel extraction kit from Qiagen. Any other phenol-free method that leaves little salt contamination is probably equivalent.

3. Repeat step 2.

4. Determine the DNA concentration by spectrophotometry.

5. Dilute the DNA to 1-5 ng/^L and check the concentration using comparative ethidium bromide staining with DNA standards of known concentration.

6. Filter the DNA solution through a small 0.22-^m Millipore filter attached to a syringe.

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