Precise genome editing by homologous recombination.

Simple and efficient methods are presented for creating precise modifications of the zebrafish genome. Edited alleles are generated by homologous recombination between the host genome and double-stranded DNA (dsDNA) donor molecules, stimulated by the induction of double-strand breaks at targeted loci in the host genome. Because several kilobase-long tracts of sequence can be exchanged, multiple genome modifications can be generated simultaneously at a single locus. Methods are described for creating: (1) alleles with simple sequence changes or in-frame additions, (2) knockin/knockout alleles that express a reporter protein from an endogenous locus, and (3) conditional alleles in which exons are flanked by recombinogenic loxP sites. Significantly, our approach to genome editing allows the incorporation of a linked reporter gene into the donor sequences so that successfully edited alleles can be identified by virtue of expression of the reporter. Factors affecting the efficiency of genome editing are discussed, including the finding that dsDNA products of I-SceI meganuclease enzyme digestion are particularly effective as donor molecules for gene-editing events. Reagents and procedures are described for accomplishing efficient genome editing in the zebrafish.

MRP class of human ATP binding cassette (ABC) transporters: historical background and new research directions.

1. The adenosine triphosphate (ATP) binding cassette (ABC) transporters form one of the largest protein families encoded in the human genome, and more than 48 genes encoding human ABC transporters have been identified and sequenced. It has been reported that mutations of ABC protein genes are causative in several genetic disorders in humans. 2. Many human ABC transporters are involved in membrane transport of drugs, xenobiotics, endogenous substances or ions, thereby exhibiting a wide spectrum of biological functions. According to the new nomenclature of human ABC transporter genes, the 'ABCC' gene sub-family comprises three classes involving multidrug resistance-associated proteins (MRPs), sulfonylurea receptors (SURs), and a cystic fibrosis transmembrane conductance regulator (CFTR). 3. Molecular cloning studies have identified a total of ten members of the human MRP class including ABCC11, ABCC12, and ABCC13 (pseudo-gene) that have recently been characterized. 4. This review addresses the historical background and discovery of the ATP-driven xenobiotic export pumps (GS-X pumps) encoded by MRP genes, biological functions of ABC transporters belonging to the MRP class, and regulation of gene expression of MRPs by oxidative stress.

A biomechanical, radiologic, and clinical comparison of outcome after multilevel cervical laminectomy or laminoplasty in the rabbit.

STUDY DESIGN: A rabbit model was used to compare clinical outcome, radiographic changes, and biomechanical flexibility after cervical laminectomy and open-door laminoplasty. OBJECTIVE: This study tested the hypothesis that radiographic changes and biomechanical flexibility could explain the differences in clinical outcome after cervical laminectomy and laminoplasty. SUMMARY OF BACKGROUND DATA: Although multilevel cervical laminoplasty is thought to have advantages over cervical laminectomy, clinical outcome studies have been contradictory, and no experimental study has examined the possible mechanisms for the differences after healing. METHODS: Twenty-four New Zealand White rabbits were randomized into four groups: normal, sham, C3-C6 wide laminectomy, and C3-C6 open-door laminoplasty. Clinical, radiographic, and biomechanical data were collected and compared up to 3 months after surgery. RESULTS: Laminectomy had a statistically significant poorer clinical outcome when compared with laminoplasty after 3 months of healing. Radiologic analysis showed statistically significant angular deformity in the laminectomy group compared with laminoplasty and control groups at 3 months. In contrast, biomechanical measures of flexibility, neutral zone, and range of motion showed only small differences between any of the groups at any time. CONCLUSIONS: The presence of deformity, and not a change in flexibility, is responsible for the differences in clinical outcome observed after laminectomy compared with laminoplasty in this model.

Clinical outcome scales for use in a rabbit model of cervical myelopathy.

This study determined the ability of an upper extremity Tarlov scale, a lower extremity Tarlov scale, and the Durham scale to predict the development of myelopathy and the likelihood of survival in a rabbit model of surgical treatments for cervical spondylotic myelopathy. Forty-eight rabbits were evaluated using the scales after cervical spinal surgery. Logistic regression analysis revealed that all three scales could predict the occurrence of myelopathy. However, only the Durham and lower extremity Tarlov scales also predicted the likelihood of survival. The Durham scale is offered as a useful predictor of myelopathy and survival in an animal model of surgical treatments for cervical spondylotic myelopathy. The lower extremity Tarlov scale is also a useful predictor of outcome; however, the upper extremity Tarlov scale is not recommended.

Strength and stability of posterior lumbar interbody fusion. Comparison of titanium fiber mesh implant and tricortical bone graft.

STUDY DESIGN: A paired comparison was done of the bending flexibility and compression strength of tricortical bone graft and titanium fiber mesh implants in a human cadaver model of posterior lumbar interbody fusion. OBJECTIVES: To test the hypothesis that a titanium fiber mesh implant and a tricortical bone graft provide adequate and equal mechanical strength and stability in posterior lumbar interbody fusion constructs. SUMMARY OF BACKGROUND DATA: Although studies of posterior lumbar interbody fusion constructs have been performed, the authors are unaware of any study in which the strength and stability of a titanium fiber mesh implant are compared with those of tricortical bone graft for posterior lumbar interbody fusion in the human cadaver lumbar spine. METHODS: Changes in neutral zone and range of motion were measured in a bending flexibility test before and after placement of posterior lumbar interbody fusion constructs. Tricortical bone graft and titanium fiber mesh implant construct stability than were compared in a paired analysis. The constructs than were loaded to failure to evaluate construct strength as a function of graft material and bone mineral density. RESULTS: The posterior lumbar interbody fusion procedure produced statistically significant decreases in neutral zone when compared with the intact spine. No statistically significant differences in neutral zone, range of motion, or strength were detected between the two implants. Construct strength correlated strongly with bone mineral density. CONCLUSIONS: Posterior lumbar interbody fusion procedures result in equal or improved acute stability for titanium fiber mesh implants and tricortical bone graft implants when used without additional posterior stabilization.

Transcriptional regulation of the Sex-lethal gene by helix-loop-helix proteins.

Somatic sex determination in Drosophila depends on the expression of Sex-lethal (Sxl), whose level is determined by the relative number of X chromosomes and sets of autosomes (X:A ratio). The first step in regulation of Sxl expression is transcriptional control from its early promoter and several genes encoding transcription factors of the helix-loop-helix (HLH) family such as daughterless (da), sisterless-b (sis-b), deadpan (dpn) and extramacrochaetae (emc) have been implicated. By the use of transfection assays and in vitro binding experiments, here we show that da/sis-b heterodimers bind several sites on the Sxl early promoter with different affinities and consequently tune the level of active transcription from this promoter. Interestingly, our data indicate that repression by the dpn product of da/sis-b dependent activation results from specific binding of dpn protein to a unique site within the promoter. This contrasts with the mode of emc repression, which inhibits the formation of the da/sis-b heterodimers. These results reveal the molecular mechanisms by which Sxl gene transcription is positively or negatively regulated to control somatic sex determination.

Binding of the Drosophila transformer and transformer-2 proteins to the regulatory elements of doublesex primary transcript for sex-specific RNA processing.

Sex-specific alternative processing of double-sex (dsx) precursor messenger RNA (pre-mRNA) is one of the key steps that regulates somatic sexual differentiation in Drosophila melanogaster. By transfection analyses using dsx minigene constructs, we identified six copies of the 13-nucleotide sequences TC(T/A)(T/A)C(A/G)ATCAACA in the female-specific fourth exon that act as the cis elements for the female-specific splicing of dsx pre-mRNA. UV-crosslinking experiments revealed that both female-specific transformer (tra) and transformer-2 (tra-2) products bind to the 13-nucleotide sequences of dsx pre-mRNA. These results strongly suggest that the female-specific splicing of dsx pre-mRNA is activated by binding of these proteins to the 13-nucleotide sequences.

Control of doublesex alternative splicing by transformer and transformer-2 in Drosophila.

Sex-specific alternative processing of doublesex (dsx) precursor messenger RNA (pre-mRNA) regulates somatic sexual differentiation in Drosophila melanogaster. Cotransfection analyses in which the dsx gene and the female-specific transformer (tra) and transformer-2 (tra-2) complementary DNAs were expressed in Drosophila Kc cells revealed that female-specific splicing of the dsx transcript was positively regulated by the products of the tra and tra-2 genes. Furthermore, analyses of mutant constructs of dsx showed that a portion of the female-specific exon sequence was required for regulation of dsx pre-messenger RNA splicing.

Binding of the Drosophila sex-lethal gene product to the alternative splice site of transformer primary transcript.

Somatic sexual differentiation in Drosophila melanogaster is accomplished by a hierarchy of genes of which one, Sex-lethal (Sxl), is required for the functional female-specific splicing of the transcripts of the immediately downstream regulatory gene, transformer (tra). The first exon of the tra primary transcript is spliced to one of two acceptor sites. Splicing to the upstream site yields a messenger RNA which is neither sex-specific nor functional, but that produced after splicing to the downstream acceptor site yields a functional female-specific mRNA. Here we address the question of how the Sxl gene product determines the alternative splicing of tra primary transcripts. One suggestion is that non-sex-specific splicing to the upstream acceptor is blocked in female flies by sex-specific factors, but neither the identity of the female-specific factors nor the mechanism of the blockage has been specified. We have now performed co-transfection experiments in which Sxl complementary DNA and the tra gene are expressed in Drosophila Kc cells. Moreover, we find that female Sxl-encoded protein binds specifically to the tra transcript at or near the non-sex-specific acceptor site, implying that the female Sxl gene product is the trans-acting factor that regulates the alternative splicing.

[Experimental studies of titanium fiber metal implant for spine fusion].

For use as a graft in spine fusion, a titanium fiber metal implant (TFMI) from a woven pure titanium wire 250 micron in diameter was studied for its mechanical properties and biological fixation. Compression testing showed that the failure stress and modulus of elasticity of our implant closely resembled those previously reported fiber metals. It was, furthermore found that the pore-size distribution and mechanical properties of our TMFI were less variable than those of the products from kinked-and-cut wires. Repetitive-loading test showed a low permanent strain rate for TFMI, even with loads much greater than the failure stress of the vertebral body. Histologically and biomechanically, sufficient biological fixation was observed in the TMFI implanted lumbar vertebrae of adult mongrel dogs. According to these favorable results, TFMI could be an excellent biomaterial for spine fusion.