Patricia Giraldo

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.

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.

The potential benefits of insulators on heterologous constructs in transgenic animals.

Transgenes can display position effects, shown as variegated, ectopic, uncontrolled and even silenced expression. These undesired side effects result in irreproducible and non-systematic expression patterns in transgenic animals. The use of artificial-chromosome type constructs has been shown to overcome these position effects (Giraldo & Montoliu, 2001). However, in a large proportion of experiments with transgenic animals, constructs are made by fusing the coding region of a given gene under the control of heterologous promoter and regulatory sequences, with the aim that the expression pattern of the heterologous gene would be faithfully reproduced by the transgenic chimeric construct. In this context, the lack of endogenous regulatory sequences that are required for establishing a proper expression domain, often result in poorly controlled and suboptimally expressed transgenes (Montoliu, 2002). Insulators have been identified in a limited number of genes from various organisms (West et al., 2002). These regulatory elements are characterised by (1) their capacity to establish genomic barriers, protecting DNA sequences from the spreading of neighbouring heterochromatin, and (2) their potential to interfere with the activity from distally located enhancers. These two properties make them ideal compounds of any transgenic construct design and, accordingly, their inclusion has been considered and recommended for biotechnological, biomedical and biological purposes (Houdebine, 2000; Montoliu, 2002). The first insulators from animal genomes were described in Drosophila and subsequently have been

Tyrosinase gene expression is not detected in mouse brain outside the retinal pigment epithelium cells

Tyrosinase is the rate‐limiting enzyme for melanin synthesis. Its gene is expressed in two cell types: melanocytes, derived from migrating neural crest cells, and, in the CNS, retinal pigment epithelium cells, derived from the optic cup. Its absence from the eye results in profound pathway selection errors of optic fibres at the chiasm and, hence, it has been implicated as a developmental regulator of CNS pathway selection. Recently, it has been proposed that tyrosinase can also be expressed in the developing and adult brain, although the methods used were indirect. Its presence in the brain could be very significant in terms of a potentially wider role in pathway finding. Here, we have evaluated the presence of tyrosinase expression in mouse developing, perinatal and adult brain by in situ hybridization in whole‐mount embryos and histological sections and by real‐time reverse transcription–polymerase chain reaction. We find no evidence for tyrosinase gene expression in the CNS outside the retinal pigment epithelium cells.

Generation of Yeast Artificial Chromosome Transgenic Mice by Intracytoplasmic Sperm Injection

Genomic-type transgenes are usually expressed in appropriate spatial- and temporal-specific manners. The largest genomic transgenes can be prepared using yeast artificial chromosomes (YACs). Normally, YAC transgenic mice are produced by standard pro-nuclear microinjection, although other methods, involving the use of embryonic stem (ES) cells, have been also devised. To overcome the difficulty and time extension of ES cell-type approaches and to improve the rather usual low efficiency of YAC DNA transgenesis by pronuclear microinjection, that is mostly dependent on the YAC DNA quality of samples, we have devised an updated intracytoplasmic sperm injection (ICSI) method for the stable incorporation of YACs into the germ line of mice. DNA transgenesis efficiencies achieved are often 10 times greater than those usually obtained by standard microinjection, thus enabling the identification of either more transgenic founder animals and the use of reduced numbers of individuals in animal experimentation.

The use of yeast artificial chromosomes in transgenic animals: expression studies of the tyrosinase gene in transgenic mice

Variegation and inherited somatic mosaicism has been observed in transgenic mice carrying yeast artificial chromosomes (YACs) in which a DNAse I hypersensitive site (HS) located -12 kb upstream of the mouse tyrosinase gene had been deleted. At present, we are generating new transgenic animals with minor deletions of the HS.