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View the protocol, "Genome Modification by Inclusion of loxP Transgene Sequences," at BioSupplynet.com
Kazuaki Ohtsubo and Jamey D. Marth
This protocol was adapted from "Conditional Mutagenesis of the Genome Using Site-Specific DNA Recombination," Chapter 60, in Gene Transfer: Delivery and Expression of DNA and RNA (eds. Friedmann and Rossi). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2007.
ABSTRACT
We describe here a procedure for introducing loxP sites into the mammalian genome. A typical gene-targeting approach to create a conditional null allele is presented, in which the initial placement of loxP sites is not deleterious to allele function. This can be modified to include knock-ins of point mutations, with such mutations flanked by loxP sites that can then be recombined by Cre expression. The choice of sequence or regulatory element to modify is dependent on the experimental design. Consideration must be given to the possible production of truncated and altered gene sequence products, or otherwise aberrantly functioning alleles. |
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Products Available for this Protocol
| Protocol Material Description |
Product # |
Product Name |
Add to Cart |
| Dimethylsulfoxide | D2650 | Dimethyl sulfoxide, liquid (temperature dependent), ≥99.7%, Hybri-Max™, sterile-filtered, hybridoma tested |  |
| Dulbecco’s modified Eagle’s medium (DMEM) | D6046 | Dulbecco’s Modified Eagle’s Medium - low glucose, With 1000 mg/L glucose, L-glutamine, and sodium bicarbonate, liquid, sterile-filtered, cell culture tested | |
| Fetal bovine serum (FBS) | F2442 | Fetal Bovine Serum, USA origin, sterile-filtered, cell culture tested, hybridoma tested | |
| MEM nonessential amino acids | M7145 | MEM Non-essential Amino Acid Solution (100×) , liquid, without
L-glutamine, sterile-filtered, cell culture tested | |
| Sodium pyruvate | P5280 | Sodium pyruvate, cell culture tested, insect cell culture tested,
≥99%, powder | |
| β-mercaptoethanol | M3148 | 2-Mercaptoethanol, for molecular biology, ≥98% (GC/titration) | |
| 10,000 units/ml penicillin:10,000 units/ml streptomycin:200 mM L-glutamine | G6784 | L-Glutamine-Penicillin-Streptomycin Solution Stabilized, sterile-filtered, cell culture tested | |
| NaCl | S3014 | Sodium chloride, for molecular biology, ≥98% (titration) | |
| Leukemia Inhibitory Factor (LIF) | L5158 | Leukemia Inhibitory Factor from mouse, recombinant, expressed in Escherichia coli, 10 µg/ml, buffered aqueous solution, cell culture tested | |
| Ethanol | E7148 | Ethanol, for molecular biology, 190 proof | |
| G418 | A8601 | G 418 disulfate salt, Biotechnology Performance Certified | |
| Gelatin (Type A, from porcine skin) | G2500 | Gelatin from porcine skin, Type A | |
| Mineral oil | M8410 | Mineral oil, mouse embryo tested, light oil (neat) | |
| Oligonucleotides — Innovative line of custom oligonucleotides for your research |
| PBS | P5368 | Phosphate buffered saline, pH 7.4, Biotechnology Performance Certified | |
| Proteinase K | P6556 | Proteinase K from Tritirachium album, lyophilized powder, ≥30 units/mg protein | |
| Restriction Endonucleases and Buffers |
| EDTA | E5134 | Ethylenediaminetetraacetic acid disodium salt dihydrate, for molecular biology, ≥99% | |
| Sarkosyl | L5000 | N-Lauroylsarcosine, neat, ≥95% | |
| Tris-Cl | T5941 | Trizma® hydrochloride, for molecular biology, Biotechnology Performance Certified, cell culture tested, ≥99.0% (titration) | |
| Trypsin-EDTA | T3924 | Trypsin-EDTA solution, 1 ×, 0.5 g porcine trypsin and 0.2 g EDTA • 4Na per liter of Hanks' Balanced Salt Solution with phenol red., sterile-filtered, cell culture tested | |
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The Sigma-Aldrich products listed for this specific protocol were selected either to match or to supplement the products listed within the actual protocol. The products/reagents from Sigma-Aldrich have been qualified for usage, but may not have been validated for this specific application. Please refer to the detailed product description on the usage of specific products of interest.
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