Molecular Techniques and Methods

Gene Targeting by Homologous Recombination

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Homologous replacement is versatile, in that sequences can be directly targeted and altered, inserted, or deleted. In small-fragment homologous replacement, the desired change in the target sequence was flanked on either side with sequence homologous to the endogenous sequence. After entering the cell, the fragment paired its homolog and replaced the endogenous sequence with the exogenously introduced fragment sequence. The small-fragment homologous replacement technique can be used to alter any genomic or recombinant DNA, as long as the sequence has been defined. However, the success of such site-directed mutagenesis will depend on the system to be altered, as well as other factors including the length of sequence homology contained in the fragment, the degree of nonhomology, the proliferative transcriptional state of the gene to be altered, and how the fragment is delivered into cells.




MATERIALS AND SOLUTIONS

10 x RecA Protein Reaction Buffer (1 ml)
100 mM Tris-acetate (pH 7.5) --------------------- 100 ul of 1 M Tris-acetate
10 mM DTT --------------------------------------- 10 ul of 1 M DTT
500 mM Na-acetate (pH 7.0) ---------------------- 167 ul of 3 M Na-acetate
Distilled H2O --------------------------------------- 723 ul


SF (Serum-Free) Medium
MEM (Eagle's Minimal Essential Medium)
2 mM L-glutamine


DOTAP (Cationic Lipid Transfection-Reagent)


HBS (HEPES Buffered Saline, pH 7.4-7.6) (1 L)
HEPES ---------------------------------------------- 4.9 g
NaCl ------------------------------------------------ 7.13 g
KCl ------------------------------------------------- 0.2 g
Glucose --------------------------------------------- 1.7 g
Na2HPO4.7H2O ------------------------------------ 3.75 g
0.5% Phenol red ------------------------------------ 0.25 ml
Add distilled H2O to make a final volume of ------- 1 L




PROCEDURES

Tissue Culture

1. Grow cell lines under humidified conditions in a 3-5% CO2 atmosphere at 37oC.


Preparation of Small DNA Fragments for Homologous Recombination

2. Generate 100-300 ug small DNA fragments (300-800 bp), flanked on either side with sequence homologous to the endogenous sequence, by PCR.

3. Do phenol: chloroform extraction.
Do chloroform: IAA extraction.

4. Add 1/10th volume of 3 M Na-acetate (pH 7.0) and 2 x the volume of 100% ethanol.

5. Precipitate the DNA fragment at -20oC for 1 hour.

6. Centrifuge 10 min at 13,000 rpm to pellet the DNA.

7. Wash the DNA pellet with 70% ethanol and air-dry.

8. Resuspend the DNA pellet in pyrogen (lipopolysaccharides)-free water at a concentration of 1 ug/ ul.


Transfection and Analysis

9. Denature 50 ul DNA fragment by boiling for 10 min.

10. Place immediately in an ice-water bath.

11. Add the following components to coat the denatured-DNA fragment with RecA protein:

Denatured DNA
5 ug
RecA protein
200 ug
100 mM ATP
3.3 ul
100 mM Mg-acetate
1.34 ul
10 x RecA Protein Reaction Buffer
6.7 ul
Add distilled H2O to make a final volume of
67 ul


12. Incubated 10 min at 37oC.

13. Add 7 ul 200 mM Mg-acetate to stop the RecA coating reaction.

  • Under these conditions, the ssDNA fragments are coated with RecA protein at a molar ratio of 3 bases per 1 RecA protein molecule.

    14. Immediately place the RecA protein-coated DNA fragments on ice.

    15. Add 95 ul of SF medium to the 5 ul of denatured, RecA-coated DNA.

    16. Suspend a 35 ul aliquot of DOTAP in 65 ul SF medium.

    17. Add the the lipid mixture from atep 16 dropwise to the DNA mixture from step 15.

    18. Keep at room temperature for 15-30 min.
  • The complex-forming reaction is carried out in polystyrene tubes.

    19. Plate 1 x 105-6 cells into a T25 flask the day before transfection.
  • Replace with fresh medium several hours before transfection.

    20. Immediately before transfection, remove the medium and wash the cells with HBS.

    21. Add 1.5 ml SF media to the flask.

    22. Add the DNA-DOTAP complex to the media in the flask.

    23. Incubate cells for 4 hour in a 3-5% CO2 atmosphere at 37oC.

    24. Remove the transfection solution.

    25. Wash the cells with HBS. Repeat 2 times.

    26. Feed the cells with 3.5 ml complete MEM or DMEH (Dulbecco's Modified Eagle's/Ham's F12) medium .

    27. Isolate DNA and RNA from transfected cells.

    28.Do PCR, RT-PCR and southern blot hybrdization.

    29. After southern blot hybridization, expose the membranes to X-ray film.

    30. Analyze autoradiographs by scanning with a Scanning Densitometer to determine the efficiency of homologous replacement.

    31. Determine the relative efficiency of hybridization of the probes by comparing the intensity of transmission after hybridization to PCR-fragments between wild type and mutants.




    NOTES

  • The DNA fragment was first denatured prior to transfection, to reduce the number of random incorporation events. Theoretically, if the fragment is single-stranded, it will preferentially pair with homologous sequences, thus greatly inhibiting the ability to integrate randomly. Double-stranded DNA is able to more readily integrate into the genomic DNA at random sites. Such random integration can result in insertional mutagenesis and the obstruction of gene expression or cell death.

  • Because much of the analysis of small-fragment homologous replacement is performed using PCR, it is important to control for artifacts associated with PCR to avoid false positive results. Appropriate control samples must always be run, along with any experimental samples, i.e. a positive control, a negative control, and water. In addition, RNA can be treated with DNase to eliminate nonspecific amplification.

  • The correction of the sequence should be a permanent correction. However, it is possible that DNA repair machinery will correct the exogenously introduced DNA after replacement. In addition, if the transcribed strand is transiently replaced by the exogenous DNA, a temporary correction would be detected. The mismatch would be transiently transcribed and expressed as functional protein. However, the sequence would eventually revert to the original endogenous form.




    KIT INFORMATION




    REFERENCES

  • Capecchi, MR (1989) Altering the genome by homologous recombination. Science 224: 1288-1292.

  • Capecchi, MR (1989) New mouse genetics: altering the genome by gene targeting. Trends Genet. 5: 70-76.

  • Fujitani, Y, Yamamoto, K, Kobayashi, I (1995) Dependence of frequency of homologous recombination on the homology length. Genetics 140: 797-809.

  • Hunger-Bertling, K, Harrer, P, Bertling, W (1990) Short DNA fragments induce site specific recombination in mammalian cells. Mol. Cell Biochem. 92: 107-116.

  • Morrison, C, Wagner, E (1996) Extrachromosomal recombination occurs efficiently in cells defective in various DNA repair systems. Nucleic Acids Res.: 24,2053-2058.


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