Lluís Montoliu

Size matters: use of YACs, BACs and PACs in transgenic animals.

In 1993, several groups, working independently, reported the successful generation of transgenic mice with yeast artificial chromosomes (YACs) using standard techniques. The transfer of these large fragments of cloned genomic DNA correlated with optimal expression levels of the transgenes, irrespective of their location in the host genome. Thereafter, other groups confirmed the advantages of YAC transgenesis and position-independent and copy number-dependent transgene expression were demonstrated in most cases. The transfer of YACs to the germ line of mice has become popular in many transgenic facilities to guarantee faithful expression of transgenes. This technique was rapidly exported to livestock and soon transgenic rabbits, pigs and other mammals were produced with YACs. Transgenic animals were also produced with bacterial or P1-derived artificial chromosomes (BACs/PACs) with similar success. The use of YACs, BACs and PACs in transgenesis has allowed the discovery of new genes by complementation of mutations, the identification of key regulatory sequences within genomic loci that are crucial for the proper expression of genes and the design of improved animal models of human genetic diseases. Transgenesis with artificial chromosomes has proven useful in a variety of biological, medical and biotechnological applications and is considered a major breakthrough in the generation of transgenic animals. In this report, we will review the recent history of YAC/BAC/PAC-transgenic animals indicating their benefits and the potential problems associated with them. In this new era of genomics, the generation and analysis of transgenic animals carrying artificial chromosome-type transgenes will be fundamental to functionally identify and understand the role of new genes, included within large pieces of genomes, by direct complementation of mutations or by observation of their phenotypic consequences.

Generation and phenotypic analysis of sigma receptor type I (σ1) knockout mice

Sigma (σ) sites are a type of nonopiate receptor whose role has been associated with several behaviours, including anxiety, depression, analgesia, learning processes and psychosis. Although there are several known σ receptor types, only the type I receptor (σ1) has been cloned. To uncover the in vivo relevance of σ‐receptors, we have generated knockout mice for σ1. Despite the broad expression pattern found for the σ1‐gene, homozygous mutant mice are viable, fertile and do not display any overt phenotype, compared with their wild‐type litter‐mates, in mixed genetic backgrounds. However, a significant decrease in the hypermotility response has been measured in knockout mice upon challenge with (+)SKF‐10 047, in agreement with the involvement of σ1‐receptors in the induction of psychostimulant actions. The activity of σ2‐receptors seems to be unaffected in σ1‐mutant mice. These knockout mice could contribute to better understand the in vivo role of σ‐receptors.

Structure and expression of the lignin O-methyltransferase gene from Zea mays L.

The isolation and characterization of cDNA and homologous genomic clones encoding the lignin O-methyltransferase (OMT) from maize is reported. The cDNA clone has been isolated by differential screening of maize root cDNA library. Southern analysis indicates that a single gene codes for this protein. The genomic sequence contains a single 916 bp intron. The deduced protein sequence from DNA shares significant homology with the recently reported lignin-bispecific caffeic acid/5-hydroxyferulic OMTs from alfalfa and aspen. It also shares homology with OMTs from bovine pineal glands and a purple non-sulfur photosynthetic bacterium. The mRNA of this gene is present at different levels in distinct organs of the plant with the highest accumulation detected in the elongation zone of roots. Bacterial extracts from clones containing the maize OMT cDNA show an activity in methylation of caffeic acid to ferulic acid comparable to that existing in the plant extracts. These results indicate that the described gene encodes the caffeic acid 3-O-methyltransferase (COMT) involved in the lignin biosynthesis of maize.

A cell-specific enhancer far upstream of the mouse tyrosinase gene confers high level and copy number-related expression in transgenic mice

The tyrosinase gene encodes the key enzyme of melanin production and is tightly regulated during development. A yeast artificial chromosome covering the mouse tyrosinase gene has been shown to rescue completely the albino phenotype of recipient mouse strains, conferring copy number‐dependent, position‐independent expression. To investigate the presence of cis‐acting regulatory elements responsible for the appropriate expression of the tyrosinase gene, DNase I hypersensitive site mapping was performed. A melanoma cell‐specific DNase I hypersensitive site was identified at ‐12 kb upstream of the tyrosinase gene. Functional analysis of the corresponding cis‐acting element in transgenic mice and transient transfection assays revealed properties of a strong cell‐specific enhancer. RNA expression levels of the transgene correlate with copy number, which is reflected in coat colour and eye pigmentation of transgenic mice. Full enhancer activity in transient transfections is obtained with a minimal sequence of 200 bp. Protein binding analysis reveals the presence of a melanoma cell‐specific complex which might contribute to the faithful expression of the tyrosinase gene.

Genetic polymorphisms among C57BL/6 mouse inbred strains

Mice from the inbred C57BL/6 strain have been commonly used for the generation and analysis of transgenic and knockout animal models. However, several C57BL/6 substrains exist, and these are genetically and phenotypically different. In addition, each of these substrains can be purchased from different animal providers and, in some cases, they have maintained their breeding stocks separated for a long time, allowing genetic differences to accumulate due to individual variability and genetic drift. With the aim of describing the differences in the genotype of several C57BL/6 substrains, we applied the Illumina® Mouse Medium Density Linkage Mapping panel, with 1,449 single nucleotide polymorphisms (SNPs), to individuals from ten C57BL/6-related strains: C57BL/6JArc, C57BL/6J from The Jackson Lab, C57BL/6J from Crl, C57BL6/JRccHsd, C57BL/6JOlaHsd, C57BL/6JBomTac, B6(Cg)-Tyrc−2j/J, C57BL/6NCrl, C57BL/6NHsd and C57BL/6NTac. Twelve SNPs were found informative to discriminate among the mouse strains considered. Mice derived from the original C57BL/6J: C57BL/6JArc, C57BL/6J from The Jackson Lab and C57BL/6J from Crl, were indistinguishable. Similarly, all C57BL/6N substrains displayed the same genotype, whereas the additional substrains showed intermediate cases with substrain-specific polymorphisms. These results will be instrumental for the correct genetic monitoring and appropriate mouse colony handling of different transgenic and knockout mice produced in distinct C57BL/6 inbred substrains.

Zebrafish enhancer detection (ZED) vector: A new tool to facilitate transgenesis and the functional analysis of cis-regulatory regions in zebrafish

The identification and characterization of the regulatory activity of genomic sequences is crucial for understanding how the information contained in genomes is translated into cellular function. The cis‐regulatory sequences control when, where, and how much genes are transcribed and can activate (enhancers) or repress (silencers) gene expression. Here, we describe a novel Tol2 transposon‐based vector for assessing enhancer activity in the zebrafish (Danio rerio). This Zebrafish Enhancer Detector (ZED) vector harbors several key improvements, among them a sensitive and specific minimal promoter chosen for optimal enhancer activity detection, insulator sequences to shield the minimal promoter from position effects, and a positive control for transgenesis. Additionally, we demonstrate that highly conserved noncoding sequences homologous between humans and zebrafish largely with enhancer activity largely retain their tissue‐specific enhancer activity during vertebrate evolution. More strikingly, insulator sequences from mouse and chicken, but not conserved in zebrafish, maintain their insulator capacity when tested in this model. Developmental Dynamics 238:2409–2417, 2009.

A tandem of α-tubulin genes preferentially expressed in radicular tissues from Zea mays

The identification of a cDNA (MR19) corresponding to a maize α-tubulin and homologous genomic clones (MG19/6 and MG19/14) is described. The cDNA has been isolated by differential screening of a cDNA maize root library. We have found two α-tubulin genes in a tandem arrangement in the genomic clones, separated by approximately 1.5 kbp. One of the genes (gene I) contains an identical nucleotide sequence which corresponds to the cDNA clone. The two deduced proteins from DNA sequences are very similar (only two conservative replacements in 451 amino acids) and they share a high homology as compared with the published α-tubulin sequences from other systems and in particular with the Arabidopsis thaliana and Chlamydomonas reinhardtii sequences reported. The structure of both genes is also very similar; it includes two introns, of 1.7 kbp and 0.8 kbp respectively, in each gene and only one intron placed at a homologous position in relation to Arabidopsis thaliana genes. By using specific 3′ probes it appears that both genes are preferentially expressed in the radicular system of the plant. The α-tubulin gene family of Zea mays seems to be represented by at least 3 or 4 members.

Increasing the complexity: new genes and new types of albinism

Albinism is a rare genetic condition globally characterized by a number of specific deficits in the visual system, resulting in poor vision, in association with a variable hypopigmentation phenotype. This lack or reduction in pigment might affect the eyes, skin, and hair (oculocutaneous albinism, OCA), or only the eyes (ocular albinism, OA). In addition, there are several syndromic forms of albinism (e.g. Hermansky–Pudlak and Chediak–Higashi syndromes, HPS and CHS, respectively) in which the described hypopigmented and visual phenotypes coexist with more severe pathological alterations. Recently, a locus has been mapped to the 4q24 human chromosomal region and thus represents an additional genetic cause of OCA, termed OCA5, while the gene is eventually identified. In addition, two new genes have been identified as causing OCA when mutated: SLC24A5 and C10orf11, and hence designated as OCA6 and OCA7, respectively. This consensus review, involving all laboratories that have reported these new genes, aims to update and agree upon the current gene nomenclature and types of albinism, while providing additional insights from the function of these new genes in pigment cells.

Efficient Generation of Transgenic Mice with Intact Yeast Artificial Chromosomes by Intracytoplasmic Sperm Injection

Abstract The production of animals with large transgenes is an increasingly valuable tool in biotechnology and for genetic studies, including the characterization and manipulation of large genes and polygenic traits. In the present study, we describe an intracytoplasmic sperm injection (ICSI) method for the stable incorporation and phenotypic expression of large yeast artificial chromosomes (YAC) constructs of submegabase and megabase magnitude. By coinjecting spermatozoa and YACs into metaphase II oocytes, we were able to produce founders exhibiting germline transmission of an intact and functional transgene of 250 kilobases, carrying the mouse tyrosinase locus, used here as a reporter gene to rescue the albinism of recipient mice. More than 35% transgenesis was obtained for this YAC transgene. When compared with the pronuclear microinjection standard method, the efficiency of the ICSI-mediated YAC transfer system was significantly greater. In summary, we describe, for the first time, stable incorporation in the host genome and correct phenotypic expression of large DNA constructs mediated by ICSI.

Genome-wide CTCF distribution in vertebrates defines equivalent sites that aid the identification of disease-associated genes

Many genomic alterations associated with human diseases localize in noncoding regulatory elements located far from the promoters they regulate, making it challenging to link noncoding mutations or risk-associated variants with target genes. The range of action of a given set of enhancers is thought to be defined by insulator elements bound by the 11 zinc-finger nuclear factor CCCTC-binding protein (CTCF). Here we analyzed the genomic distribution of CTCF in various human, mouse and chicken cell types, demonstrating the existence of evolutionarily conserved CTCF-bound sites beyond mammals. These sites preferentially flank transcription factor–encoding genes, often associated with human diseases, and function as enhancer blockers in vivo, suggesting that they act as evolutionarily invariant gene boundaries. We then applied this concept to predict and functionally demonstrate that the polymorphic variants associated with multiple sclerosis located within the EVI5 gene impinge on the adjacent gene GFI1.