Preparation of Copy Standards for Southern Blot Copy Number Determination and PCR Genotyping Sensitivity
PCR screens must be designed to detect transgene DNA at the single copy level or 0.1 copy level.. Southern Blots analysis of transgenic mice need copy standards to estimate copy number. Copy standards are prepared by mixing non-transgenic tail DNA with a known amount of transgene DNA is to produce transgene copy standards.
For PCR, these standards can be used to determine the sensitivy of the PCR assay. You must obtain single copy sensitivity in your PCR prior to transgene submission. When you test DNA from potentially transgenic founder mice, run your single copy PCR test sample to ensure that your PCR assay is sensitive enough to avoid false negatives.
Southern Blots are commonly used to determine transgene copy number and the number of integration sites in transgenic founder mice.
Calculation of Copy Number Standards
Assumption: the Haploid content of a mammalian genome is 3 X 109 bp
Assumption: you have 2 micrograms of tail DNA available
Since the transgenic founder mice are hemizygous:
mass of transgene DNA = N bp transgene DNA
1 microgram genomic DNA 3 X 109 bp genomic DNA
Example: for a 5,480 bp transgene insert or plasmid
mass of transgene DNA = 5,480 bp cloned DNA or
1 micrograms genomic DNA 3 X 109 bp genomic DNA
mass of transgene DNA = (5,480 bp cloned DNA) X (1 µg genomic DNA) or
3 X 109 bp genomic DNA
mass of transgene DNA = 1.83 picograms per 1 ug genomic DNA
transgenic mice will be hemizygous for the transgene, not homozygous
1.83pg transgene should be added to 2 ug of genomic DNA
Thus, to prepare a 1 copy standard: add 1.83 pg of transgene DNA to 2 microgram tail DNA
0.1 copy 0.183 pg
10 copy 18.3 pg
50 copy 91.5 pg
100 copy 183 pg
For use as a transgene PCR standard, use 200 ng of the spiked tail DNA as a substrate in a 25 ul PCR reaction.
For use in Southern blot analysis, digest the tail DNA as you would for Southern analysis, and add the transgene insert DNA (not the entire plasmid) just before you load your gel. Remember to reserve one lane for genomic DNA only with no spike. For an example of copy standards in Southern blots, refer to Camper SA. 1987. Research applications of transgenic mice. Biotechniques 5, 638-650.
This procedure describes how to process samples for lacZ staining. Beta galactosidase is an enzyme that hydrolizes beta galactosides. The cleavage of Xgal (5-bromo-4-chloro-3-indolyl-b -galactopyranoside) results in a dark blue precipitate. A nuclear localized lacZ transgene can be used to mark transgene expressing cells unambiguously (endogenous enzyme activity is cytosolic). If desired, antibody staining can be carried out for cytosolic proteins (see Brinkmeier et al. ). Thick specimens, such as late stage mouse embryos, can be cleared by treatment with (see Turkay et al.). Background staining can be reduced by increasing the pH of the phosphate buffers used to process samples. For a review see the chapter by Saunders.
lacZ Staining Procedure for Cells and Mouse Embryos:
1. Rinse cells (or mouse embryos) once in phosphate buffer pH 7.3 at room temperature.
2. Fix cells for 5 minutes (embryos for 15-30 minutes, depending on size) at room temp.
3. Wash cells 3 times for 5 minutes (15 minutes for embryos) at room temperature.
4. Stain cells (embryos) 4 hours to overnight at 37*C depending on level of LacZ activity.
5. After staining, pour off stain, replace with wash buffer.
6. Store samples at 4*C. Staining will intensify in wash buffer at 4*C.
Notes on Staining:
The procedure doesn't have background problems in most cell types. With embryos, background staining is seen in the yolk sac of day 10 embryos and on. In addition, a thin stripe of staining is observed in the hindbrain of day 12 embryos. If background is a problem, then try increasing the pH of the phosphate buffer. Staining procedure works at pH 8.5.
Staining Procedure for Cryostat Sections:
1. Collect tissues or embryos and place in isopentane at -30 to -45*C for 30 seconds.
2. Store frozen samples at -70*C.
3. Section embryo.
4. Fix sections for 5 minutes- use glutaraldehyde fix.
5. Wash 3 times for 5 minutes.
6. Stain sections overnight at 37*C.
7. After staining, pour off and save stain, replace with wash buffer. Can counterstain with neutral red, dehydrate in ethanol and xylene and coverslip.
8. Store samples at 4*C. Staining will intensify in wash buffer at 4*C
Materials and Reagents
0.1 M phosphate buffer, pH 7.3:
115 ml 0.1 M sodium phosphate, monobasic (6.9 g in 500 ml H2O)
385 ml 0.1 M sodium phosphate, dibasic (14.2 g in 500 ml H2O)
500 ml total
Fix Solution - Always prepare fresh:
4 ml 25% glutaraldehyde
2.5 ml 100 mM EGTA, pH 7.3
0.4 ml 1 M MgCl2
173.1 ml double distilled H2O
20.0 ml 10x PBS
200.0 ml total
Wash Buffer:
0.4 ml 1 M MgCl2
2.0 ml 1% deoxycholate (may be omitted)
2.0 ml 2% NP-40
195.6 ml 0.1 M sodium phosphate, pH 7.3
200.0 ml total
X-gal stock:
250 mg X-gal (5-bromo-4-chloro-3-indolyl-b -galactopyranoside) (Sigma B4252)
10 ml dimethyl formamid (Sigma D4551)
X-gal stain - Make fresh:
2.0 ml 25 mg/ml X-gal stock
0.106 g potassium ferrocyanide (Sigma P-9387)
0.082 g potassium ferricyanide (Sigma P-8131)
48.0 ml wash buffer
50.0 ml total
