Genotyping

1. Obtain tail biopsies from 2 to 3 week old mice: Hold mouse firmly at base of tail with one hand, with the other cut off 0.5 to 1.0 cm of the tail tip with a scalpel or single edge razor blade.

2. Add 600 microliters of TNES and 35 microliters Proteinase K (10 mg/ml).

3. Incubate overnight (8-24 hr) at 55°C.

4. Add 166.7 microliters 6M NaCl. Shake vigorously for 15 sec.

5. Microfuge (12,000-14,000 xg) for 5 min at room temp.

6. Remove supernatant to new tube and add 2 volumes cold 95% ethanol (EtOH).

7. Spool precipitated DNA with closed end capillary tube.

8. Rinse DNA pellet with 70% EtOH (dipping in a tube filled with 70% EtOH works well) and allow to air dry 5-10 min.

9. Resuspend in 100-500 microliters of TE (10mM Tris, pH 8.0, 1mM EDTA). Volume depends empirically on pellet size.

10. Heat at 65°C for 10 minutes to aid dissolution of DNA.

11. Quantitate DNA and store at 4°C until needed.

TNES

10 mM Tris, pH 7.5
400 mM NaCl
100 mM EDTA
0.6% SDS

“6M” NaCl
Saturated salt solution stored at 37°C

The reference for the tail DNA preparation is:

Miller SA. Dykes DD. Polesky HF. 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Research. 16(3):1215.

Preparation of Copy Standards for PCR Genotyping Sensitivity and Southern Blot Copy Number Determination

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.
Download a pdf file illustrating Southern Blot analysis of transgenic founders.

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 as described: genotyping transgenic mice.

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.Click here for more review articles.

This primer pair is used to amplify the endogenous mouse beta-globin gene. Amplification will thus demonstrate that the DNA sample is a good substrate for PCR .

5′ CCA ATC TGC TCA CAC AGG ATA GAG AGG GCA GG 3′ 32 mer

5′ CCT TGA GGC TGT CCA AGT GAT TCA GGC CAT CG 3′ 32 mer

Size of expected amplification product is 494 base pairs.

Thermal cycler profile:

94°C 30 seconds
60°C 90 seconds
72°C 120 seconds

35 cycles

72°C 10 min
4°C Hold

Beta-Globin Sequence Source (Genbank Accession Number J00413):
Konkel DA, Tilghman SM, Leder P. 1978. The Sequence of the Chromosomal Mouse beta-globin Major Gene: Homologies in Capping, Splicing and Poly(A) Sites. Cell 15:1125-1132.

This primer pair is used to genotype transgenic mice that carry the beta galactosidase (lacZ) reporter gene. The primers recognize sequences within the beta galactosidase gene.

5' TTC ACT GGC CGT CGT TTT ACA ACG TCG TGA  3' 30mer

5' ATG TGA GCG AGT AAC AAC CCG TCG GAT TCT  3' 30mer

Size of expected amplification product is 364 bp.

Thermal cycler profile:

94°C 60 sec
72°CC 120 sec

30 cycles

72°C 10 min
4°C Hold

This primer pair is used test DNA for the presence of the neomycin gene.
The primers recognize sequences within the neo^r cassette of gene targeting vectors.

5′ AGG ATC TCC TGT CAT CTC ACC TTG CTC CTG 3′ 30mer

5′ AAG AAC TCG TCA AGA AGG CGA TAG AAG GCG 3′ 30mer

Size of expected amplification product is 492 base pairs.

Thermal cycler profile:

94°C 30 sec
72°C 120 sec

30 cycles

72°C 10 min
4°C Hold

This primer pair is used to amplify the endogenous rat prolactin gene. Amplification will thus demonstrate that the DNA sample is a good substrate for PCR .

5′ GCT TCT GAG CAA TGA CAC CA 3′ 18 mer

5′ ATT CCA GGA GTG CAC CAA AC 3′ 18 mer

Size of expected amplification product is 391 base pairs.

Thermal cycler profile:

1. 95°C for 45 seconds
2. 95°C for 45 seconds
3. 55°C for 1 minute
4. 72°C for 30 seconds
5. Go To #2 30 times
6. 72°C for 10 minutes
7. Hold at 4°C

Rat Prolactin Sequence Source (Genbank Accession Number AH002235):
Gubbins EJ, Maurer RA, Lagrimini M, Erwin CR, Donelson JE. Structure of the rat prolactin gene. J Biol Chem. 1980 Sep 25;255(18):8655-62.