Biomedical subjects

Andreas F Kolb

Publications and source records attributed to Andreas F Kolb.

8 recordsLinked to original sources

PubMed2006

A virus-neutralising antibody is not cytotoxic in vitro.

Hybridoma cell lines are characterized by a preferential loss of the heavy chain gene. This observation has led to the theory that the immunoglobulin heavy chain possesses an intrinsic cytotoxic activity in some cell types. We have generated transgenic mice expressing the heavy and light chain genes of the virus-neutralising antibody A1 carrying constant domains of the human gamma1 and kappa isotype. Heavy chain and light chain transgenes were under trancriptional control of identical promoter regions derived from the mammary gland specific ovine beta-lactoglobulin gene. The copy number of the heavy chain transgene was consistently lower than the copy number of the light chain gene in all lines of transgenic mice. Moreover, the light chain gene was expressed in significant excess of the heavy chain gene in the lactating mammary gland in all transgenic lines. In several transgenic lines, the differences in antibody expression were greater than could be explained by the differences in transgene copy number. One potential cause of this phenotype could be a cytotoxic effect of free heavy chain protein in embryonic cells (resulting in differences in copy number) or mammary epithelial cells (resulting in differences in transgene expression). We therefore directly assessed the effect of the expression of free A1 heavy chain protein in epithelial cell lines and in murine embryonic stem cells. However, full-length A1 heavy chain mRNA and protein could be expressed transiently and stably in both epithelial and embryonic stem cells and had no detectable effect on cell viability. Taken together, these findings argue against an inherent cytotoxicity of the free A1 heavy chain protein in epithelial or embryonic cells.

Animals↗

PubMed2005

Targeted modification of mammalian genomes.

The stable and site-specific modification of mammalian genomes has a variety of applications in biomedicine and biotechnology. Here we outline two alternative approaches that can be employed to achieve this goal: homologous recombination (HR) or site-specific recombination. Homologous recombination relies on sequence similarity (or rather identity) of a piece of DNA that is introduced into a host cell and the host genome. In most cell types, the frequency of homologous recombination is markedly lower than the frequency of random integration. Especially in somatic cells, homologous recombination is an extremely rare event. However, recent strategies involving the introduction of DNA double-strand breaks, triplex forming oligonucleotides or adeno-associated virus can increase the frequency of homologous recombination. Site-specific recombination makes use of enzymes (recombinases, transposases, integrases), which catalyse DNA strand exchange between DNA molecules that have only limited sequence homology. The recognition sites of site-specific recombinases (e.g. Cre, Flp or PhiC31 integrase) are usually 30-50 bp. In contrast, retroviral integrases only require a specific dinucleotide sequence to insert the viral cDNA into the host genome. Depending on the individual enzyme, there are either innumerable or very few potential target sites for a particular integrase/recombinase in a mammalian genome. A number of strategies have been utilised successfully to alter the site-specificity of recombinases. Therefore, site-specific recombinases provide an attractive tool for the targeted modification of mammalian genomes.

Animals↗

PubMed2005

Site-directed genome modification: nucleic acid and protein modules for targeted integration and gene correction.

A variety of technological advances in recent years have made permanent genetic manipulation of an organism a technical possibility. As the details of natural biological processes for genome modification are elucidated, the enzymes catalyzing these events (transposases, recombinases, integrases and DNA repair enzymes) are being harnessed or modified for the purpose of intentional gene modification. Targeted integration and gene repair can be mediated by the DNA-targeting specificity inherent to a particular enzyme, or rely on user-designed specificities. Integration sites can be defined by using DNA base-pairing or protein-DNA interaction as a means of targeting. This review will describe recent progress in the development of 'user-targetable' systems, particularly highlighting the application of custom DNA-binding proteins or nucleic acid homology to confer specificity.

DNA Repair↗

PubMed2005

Site-directed genome modification: derivatives of DNA-modifying enzymes as targeting tools.

The modification of mammalian genomes is an important goal in gene therapy and animal transgenesis. To generate stable genetic and biochemical changes, the therapeutic genes or transgenes need to be incorporated into the host genome. Ideally, the integration of the foreign gene should occur at sites that ensure their continual expression in the absence of any unwanted side effects on cellular metabolism. In this article, we discuss the opportunities provided by natural DNA-modifying enzymes, such as transposases, recombinases and integrases, to mediate the stable integration of foreign genes into host genomes. In addition, we discuss the approaches that have been taken to improve the efficiency and to modify the site-specificity of these enzymes.

Animals↗

PubMed2005

Regulation of genes encoding proteolytic enzymes during mammary gland development.

The mammary gland undergoes extensive tissue remodelling during each lactation cycle. During pregnancy, the epithelial compartment of the gland is vastly expanded (Benaud et al. 1998). At the end of lactation the epithelial cells undergo apoptosis and adipocyte differentiation is induced (Lilla et al. 2002). Ductal and alveolar growth during puberty and pregnancy, and the involution process require the action of proteolytic enzymes (including matrix metalloproteinases, plasminogen and membrane-peptidases) and the corresponding genes are activated during these periods (Benaud et al. 1998; Alexander et al. 2001). Matrix metalloproteinases (MMP) are expressed in several cell types of the mammary gland including stromal fibroblasts (e.g., MMP3, MMP2), epithelial cells (e.g., MMP7 or MMP9), adipocytes (e.g., MMP2) and lymphoid cells (e.g., MMP9) (Crawford et al. 1996; Lund et al. 1996; Wiseman et al. 2003). A number of knock-out mice, which are deficient for individual MMP genes (e.g., MMP2, MMP3) or plasminogen, display alterations to mammary gland structure and impairment of lactation (Lund et al. 1999; Wiseman et al. 2003).

Animals↗

PubMed2002

Structure and regulation of the murine gamma-casein gene.

The murine casein locus consists of five genes, which are coordinately regulated during mammary development. The levels of casein-specific mRNAs in mammary epithelial cells increase during the second half of pregnancy and remain high during lactation. The murine gamma-casein gene, which corresponds to the alphaS2-casein gene in ruminants, was isolated from a mouse bacterial artificial chromosome (BAC) library (strain 129SV). The gene contains 14 exons, which are distributed over 14 kb of DNA sequence. The expression pattern of the murine gamma-casein gene mimics that of the neighbouring beta-casein gene in terms of developmental induction in vivo. In cell culture, both the beta- and gamma-casein promoter are synergistically induced by prolactin and glucocorticoids. Glucocorticoid induction is critically dependent on prolactin-mediated activation of STAT5 in both promoters. Several consensus STAT5 binding sites were identified in the gamma-casein promoter, some of which may have an additive effect on prolactin induction. mRNA levels of gamma- and beta-casein are similar in lactating mammary tissue. However, promoter segments derived from the gamma-casein gene are significantly less active in cell culture than comparable fragments of the beta-casein promoter. Promoter hybrids between the gamma- and beta-casein promoters revealed that the critical sequences which are responsible for the different in vitro activity are located in a short promoter proximal region.

Animals↗

PubMed2002

Engineering immunity in the mammary gland.

The major physiological function of milk is the transport of amino acids, carbohydrates, lipids, and minerals to mammalian offspring. However, milk is also a rich collection of antimicrobial substances, which provide protection against pathogenic infections. These molecules safeguard the integrity of the lactating mammary gland, but also provide protection for the suckling offspring during a time when its immune system is still immature. The protective substances can be classified into two categories: 1) nonspecific defense substances, which provide innate immunity, and 2) molecules such as antibodies, which provide adaptive immunity and are directed against specific pathogens. The antimicrobial potency of milk has not been a target for farm animal breeding in the past, and present day ruminants provide suboptimal levels of antimicrobial substances in milk. Altered breeding regimes, pharmacological intervention, and transgenesis can be utilized to improve the antimicrobial properties of milk. Such alterations of milk composition have implications for human and animal health.

Animals↗

PubMed2002

Genome engineering using site-specific recombinases.

The targeted modification of the mammalian genome has a variety of applications in research, medicine, and biotechnology. Site-specific recombinases have become significant tools in all of these areas. Conditional gene targeting using site-specific recombinases has enabled the functional analysis of genes, which cannot be inactivated in the germline. The site-specific integration of adeno-associated virus, a major gene therapy vehicle, relies on the recombinase activity of the viral rep proteins. Site-specific recombinases also allow the precise integration of open reading frames encoding pharmaceutically relevant proteins into highly active gene loci in cell lines and transgenic animals. These goals have been accomplished by using a variety of genetic strategies but only a few recombinase proteins. However, the vast repertoire of recombinases, which has recently become available as a result of large-scale sequencing projects, may provide a rich source for the development of novel strategies to precisely alter mammalian genomes.

Animals↗