![]() Ligate together loxP sites and various other DNA fragments such as homology arms, a positive selection marker such as PGKNeo, and a negative selection marker such as MC1TK. The conventionalĪpproach is to find appropriate restriction enzyme sites that are located in or near the gene. 2001), which makes it possible to inactivate a gene in a temporal-specific fashion.Ī major limitation for generating cko mice is the difficulty and time it takes to make a cko-targeting vector. ( Hayashi and McMahon 2002) or viral delivery systems such as adenovirus or lentivirus ( Shibata et al. The timing of Cre expression can also be controlled using inducible Cre expression systems By expressing Cre recombinase from a tissue-specific promoter, the gene can be inactivated Expression of Cre recombinase in mice carrying the ckoĪllele catalyzes recombination between the loxP sites and inactivates the gene. Typically, a cko allele is made by inserting loxP sites into two introns of a gene or at the opposite ends of a gene. In a tissue- or temporal-specific fashion ( Nagy 2000). This problem can be overcome by making conditional knockout mice (cko mice), which allows a gene to be inactivated In many cases, however, the completeĭeficiency of a gene leads to embryonic lethality, precluding the analysis of gene function in later developmental stages The ability to introduce virtually any mutation into the mouse genome following gene targeting in mouse embryonic stem (ES)Ĭells provides a powerful approach for elucidating gene function in the whole animal. This method should also facilitate the generation of knock-in mutations and transgene constructs, as wellĪs expedite the analysis of regulatory elements and functional domains in or near genes. Our method is fast, efficient, and reliable and makes it possible to generate cko-targeting vectors in We also describe two new Neo selection cassettes that work well in both E. coli strains, in which the proteins required for recombination are expressed from a defective temperature-sensitive λ prophage,Īnd the Cre or Flpe recombinases from an arabinose-inducible promoter. Unlike other methods that use short 45–55-bp regions of homology for recombineering, our method To introduce loxP or FRT sites into the subcloned DNA. ![]() Λ phage Red proteins, to subclone DNA from BACs into high-copy plasmids by gap repair, and together with Cre or Flpe recombinases, This method uses homologous recombination mediated by the Method for generating conditional mouse knockout (cko) mutations. Here we describe a new recombineering-based Termed recombineering, has many different uses for functional genomic studies. Into plasmids, BACs, or PACs without the need for restriction enzymes or DNA ligases. This region is not involved at the catalytic core of the enzyme, but fragmentation in this area may result in unfolding and denaturation of the monomer followed by subsequent aggregation into large, insoluble entities and the loss of enzymatic activity.Phage-based Escherichia coli homologous recombination systems have recently been developed that now make it possible to subclone or modify DNA cloned Both tendencies suggest a potential for bond stress during freeze/thaw cycling. The IBI MacVector™ program “Protein Tool Box” predicted that this region is hydrophilic, has a high surface probability and a strong tendency to interact with water. A study of this region using the published 3 dimensional x-ray crystallographic structure of l-asparaginase revealed that the C-terminal region is exposed and can interact with water. Mass spectral data and sequence studies of these fragments in conjunction with the known sequence of the molecule placed all the fragments within the last 28 C-terminal amino acids. Automated Edman sequencing of the frozen and thawed mixture confirmed the presence of fragments and contributed some sequence information. Following up on this information, mass spectrometry was used to identify the fragments as small peptides of molecular weight 615 Da, 1424 Da and 166 Da. SEC using UV, RI and light scattering detectors and SDS-PAGE indicated that the l-asparaginase molecule fragments upon exposure to repeated freezing and thawing cycles. The cause(s) for this loss of activity were investigated using multiple techniques. chrysanthemi was found to lose activity upon exposure to consecutive freeze/thaw cycles.
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