Search

La modificación del genoma humano a voluntad es una idea que ronda a los investigadores desde los años 70 del siglo pasado. Tras la aparición de las primeras técnicas de ingeniería genética y los sucesivos métodos de transgénesis que fueron desarrollándose posteriormente siempre estuvo presente el anhelo o temor de poder modificar el ADN humano. Sin embargo esto no se pudo constatar hasta 2013, con la aparición de las herramientas de edición genética CRISPR?Cas, que facilitaron y universalizaron los procedimientos de alteración genética dirigida, sobre genes específicos.

Additional file 2: Table S1. Complete list of genes encoding identified proteins bound to the X45S and X14S Alu loci obtained via enChIP-mass spectrometry in N-TERA2 cell line.

Additional file 1. Additional figures of the manuscript including supporting information.

Additional file 3: Table S3. Complete list of primers used in chIP, 3C, enchIP and CRISPR experiments.

The communication of biotechnology has played a key role in recent years. The great advances made and the speed with which new genetic editing techniques are implemented raise enormous expectations but also concerns. Good communication of the application of biotechnology in different fields – medicine, agriculture, industry – must be accompanied by a constant dialogue between scientists and society. The idea of this monographic came from the II Conference of the Association of Biotechnology Communicators (AcB in its Spanish initialism), of which I am a member. It was held in Valencia a couple of years ago, and some of the topics that aroused the most interest, debate and participation – such as the constant flow of new information about CRISPR, animal experimentation or the importance of understanding what information a DNA analysis can provide – have served as the basis for some of the documents in this issue. Others, such as the public perception of biotechnology and the importance of the use of metaphors to explain some biotechnological processes, complete this multifaceted view of communication and biotechnology.

La comunicacion es esencial en todos los ambitos de la sociedad, pero en ciencia es una de las claves ineludibles. Comunicar es compartir, mostrar, ensenar, trasladar nuestros descubrimientos, nuestras observaciones y hallazgos a nuestros colegas, y a la sociedad en general. Por eso una buena comunicacion debe siempre acompanar a la buena ciencia. Las herramientas de edicion genetica CRISPR nos permiten modificar el genoma de cualquier organismo vivo, incluida nuestra especie, a voluntad. En este articulo desarrollare diferentes aspectos comunicativos que creo son relevantes o han ocurrido durante la corta pero intensa vida de estas tijeras moleculares, asi llamadas por su capacidad de cortar la molecula de ADN de forma efectiva y muy precisa.

Genome organization into transcriptionally active domains denotes one of the first levels of gene expression regulation. Although the chromatin domain concept is generally accepted, only little is known on how domain organization impacts the regulation of differential gene expression. Insulators might hold answers to address this issue as they delimit and organize chromatin domains. We have previously identified a CTCF-dependent insulator with enhancer-blocking activity embedded in the 5' non-coding region of the chicken α-globin domain. Here, we demonstrate that this element, called the αEHS-1.4 insulator, protects a transgene against chromosomal position effects in stably transfected cell lines and transgenic mice. We found that this insulator can create a regulated chromatin environment that coincides with the onset of adult α-globin gene expression. Furthermore, such activity is in part dependent on the in vivo regulated occupancy of CTCF at the αEHS-1.4 element. Insulator function is also regulated by CTCF poly(ADP-ribosyl)ation. Our results suggest that the αEHS-1.4 insulator contributes in organizing the chromatin structure of the α-globin gene domain and prevents activation of adult α-globin gene expression at the erythroblast stage via CTCF.

Over the years many well-described techniques for the introduction of transgene DNA into host organisms have been used, including pronuclear injection, in vitro fertilization-mediated transgenesis, transfection of ES and spermatogenic cells, nuclear transfer of somatic cell nuclei, and lentiviral vectors. The application of these techniques has been limited however either by the time and effort to be executed or by their narrow efficiency with large transgenes. The greatest advantage of intracytoplasmic sperm injection (ICSI)-mediated transgenesis is precisely its ability to stably introduce large DNA molecules into the genome of host organisms with relatively high efficiency, as compared to alternative procedures. In mice, this procedure has been shown to be a reproducible method to generate transgenic offspring with a high efficiency. Recently, it proved also to be a viable method to generate transgenic rats and pigs, and as such, it is foreseen with great interest for the production of transgenic farm animals, where it would constitute an important tool for the production of recombinant proteins and livestock improvement.

Strial melanocytes are required for normal development and correct functioning of the cochlea. Hearing deficits have been reported in albino individuals from different species, although melanin appears to be not essential for normal auditory function. We have analyzed the auditory brainstem responses (ABR) of two transgenic mice: YRT2, carrying the entire mouse tyrosinase (Tyr) gene expression-domain and undistinguishable from wild-type pigmented animals; and TyrTH, non-pigmented but ectopically expressing tyrosine hydroxylase (Th) in melanocytes, which generate the precursor metabolite, L-DOPA, but not melanin. We show that young albino mice present a higher prevalence of profound sensorineural deafness and a poorer recovery of auditory thresholds after noise-exposure than transgenic mice. Hearing loss was associated with absence of cochlear melanin or its precursor metabolites and latencies of the central auditory pathway were unaltered. In summary, albino mice show impaired hearing responses during ageing and after noise damage when compared to YRT2 and TyrTH transgenic mice, which do not show the albino-associated ABR alterations. These results demonstrate that melanin precursors, such as L-DOPA, have a protective role in the mammalian cochlea in age-related and noise-induced hearing loss.

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.

Intracytoplasmic sperm injection (ICSI) of DNA-loaded sperm cells has been shown to be a valuable tool for the production of transgenic animals, especially when DNA constructs with submegabase magnitude are used. In order to optimize and to understand the mechanism of the ICSI-mediated transgenesis, we have evaluated the impact of transgene DNA concentration, transgene flanking with nuclear matrix attachment regions (MARs), and the use of recombinase A (RecA)-coated DNA on the efficiency of mouse transgenesis production by ICSI. Presented data include assays with three DNA constructs; an enhanced green fluorescent protein (EGFP) plasmid of 5.4 kb, this plasmid flanked with two MAR elements (2.3 Kb of the human beta-interferon domain boundaries), and a yeast artificial chromosome (YAC) construct of approximately 510 kb (the largest transgenic construct introduced by ICSI that we have seen reported). ICSI-mediated transgenesis was done in the B6D2 mouse strain using different concentrations for each construct. Analysis of generated data indicated that ICSI allows the use of higher DNA concentrations than the ones used for pronuclear microinjection, however, when a certain threshold is exceeded, embryo/fetal viability decrease dramatically. In addition, independently of the transgene concentration tested, transgene flanking with MAR sequences did not have a significant impact on the efficiency of this transgenesis method. Finally, we observed that although the overall efficiency of ICSI-mediated transgenesis with fresh spermatozoa and RecA-complexed DNA was similar to the one obtained with the common ICSI-mediated transgenesis approach with frozen-thawed spermatozoa and RecA free DNA, this method was not as efficient in maintaining a low frequency of founder animal mosaicism, suggesting that different mechanisms of transgene integration might result from each procedure.

The retinal pigment epithelium (RPE) plays a key role in regulating retinal development. The critical enzyme in pigment production is tyrosinase. Transgenic mice with a tyrosinase construct where the locus control region was deleted (YRT4) display a variegated phenotype of tyrosinase expression. Their central retina is largely pigment free, whereas more peripheral regions are heavily pigmented. We have used this model to ask whether the influence of pigmented RPE over the retina during development is fundamentally governed by local interactions or is global. Our data show that YRT4 eyes have intermediate melanin content and relatively low tyrosinase activity compared with wild-type and albino animals. Rod counts are comparable to those in pigmented mice in peripheral regions but similar to those in albinos centrally. Anterograde labelling of retinal pathways demonstrates the presence of relatively normal ipsilateral chiasmatic projection in YRT4 mice, comparable with that in pigmented animals and consistent with the peripheral pigmented origin of this pathway. Examination of cellular proliferation levels during retinal development reveals that YRT4 mice display an extended period of mitosis, similar to that found in albinos. Hence, our results show that the regulatory influence of the RPE over the developing retina depends on localized interactions between these tissues.

Tyrosinase is the rate-limiting enzyme in melanin biosynthesis. It is an N-glycosylated, copper-containing transmembrane protein, whose post-translational processing involves intracytoplasmic movement from the endoplasmic reticulum to the Golgi and, eventually, to the melanosome. The expression of the tyrosinase (Tyr) gene is controlled by several regulatory regions including a locus control region (LCR) located 15 kb upstream from the promoter region. The extreme dilution mottled mutant mice (Tyrc-em) arose spontaneously at the MRC Institute in Harwell (United Kingdom) from a chinchilla-mottled mutant (Tyrc-m) stock, whose molecular basis corresponds to a rearrangement of 5'-upstream regulatory sequences including the LCR of the Tyr gene. Tyrc-em mice display a variegated pigmentation pattern in coat and eyes, in agreement with the LCR translocation, but also show a generalized hypopigmented phenotype, not seen in Tyrc-m mice. Genomic analyses of Tyrc-em mice showed a C1220T nucleotide substitution within the Tyr encoding region, resulting in a T373I amino acid change, which abolishes an N-glycosylation sequon located in the second metal ion binding site of the enzyme. Tyrosinase from Tyrc-em displayed a reduced enzymatic activity in vivo and in vitro, compared with wild-type enzyme. Deglycosylation studies showed that the mutant protein has an abnormal glycosylation pattern and is partially retained in the endoplasmic reticulum. We conclude that the phenotype of the extreme dilution mottled mouse mutant is caused by a combination of coding and noncoding genomic alterations resulting in several abnormalities that include suboptimal gene expression, abnormal protein processing, and reduced enzymatic activity.

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 generation and analysis of transgenic mice has become an important tool to progress our understanding of human and mouse gene function and its association with human genetic diseases. Animal models, based on genetically modified mice, both standard transgenic and knock-out animals, are increasingly being used worldwide. Monitoring of transgenic mouse production and transgenic mouse colonies is required to efficiently manage the resources that are available. Here, I describe three independent FileMaker databases (transgenics, mymouse and cages) that have been developed to track the generation of transgenic mice, the organisation of transgenic mouse colonies and the distribution of mice in cages. These three databases are freely available for academic use.