Germ Cells Land a Knockout Punch

Since the discovery that, with careful attention and manipulation, stem cells could be maintained in culture, one of the holy grails of transgenic research has been the ability to use spermatogonial stem cells to create transgenic animals. The advantage of these cells is that they give researchers direct access to the germ-line, since spermatogonial stem cells proliferate throughout the lifetime of the animal and continually gives rise to germ cells carrying the relevant transgene. Unfortunately, numerous hurdles have confounded this work, including difficulties in detecting, isolating, culturing, and expanding primary spermatogonial stem cells. However, these barriers have fallen one by one, resulting in a recent paper by a Japanese group who demonstrate that a knockout mouse can be produced by the manipulation of tissue-specific stem cells. Kanatsu-Shinohara et al., using their previously published culture technique for spermatogonial stem cells, were able to build on their 2005 work in which regular transgenic animals were produced, now creating occludin gene knockout mice. Utilizing either a gene trap vector or a gene targeting vector, cultured stem cells were transfected, and antibiotic-resistant clones were expanded. Following transfer into the testes of infertile mice, normal spermatogenesis was observed and more than one-third of the mice became fertile within four months. The transgene introduced was detected in approximately two-thirds of all offspring fathered by these mice, and mutant second generation offspring were shown to be deficient in occludin mRNA, both of which confirmed targeted knockout of the desired gene. Although the final success rate is still very low using this technique, it shows great promise and could have significant ramifications in the field of both transgenic animal research and gene therapy. Particularly intriguing is the possibility of its application to produce transgenic animals in species that have historically been recalcitrant to genetic manipulation using standard techniques such as nuclear transfer. –SS

-Kanatsu-Shinohara et al. 2006. Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells. Proceedings of the National Academy of Sciences 103:8018–8023.

Putting the GREM in Gremlin

Following sequencing of the human genome, a surprising find was the volume—over one-third of the total genome—represented by transposon DNA. The existence of this specific subset of so-called “junk DNA” is the work of jumping retroelements (REs), mobile genetic sequences that replicate via an RNA intermediate. When attempting to identify REs that have been co-opted as promoter elements, current techniques do not adequately distinguish between these sequences and elements without promoter functions (i.e., that are part of a larger transcript regulated by a different, upstream promoter). To tackle this shortcoming, Buzdin et al., developed a methodology they call Genomic Repeat Expression Monitor, or GREM. The technique is based on a simple premise, namely that the 5′ ends of expressed genes can be used to tag repetitive sequences that are acting as the promoters for those transcripts, marking them for isolation and future characterization. To achieve this, a library consisting of the 5′-ends of cDNAs was hybridized to a pool of repetitive sequences isolated from the entire genome. The resulting hybrid molecules were then isolated and cloned to yield a set of sequences that can be used to tag transcriptionally active repeat elements. Since the total number of each tag is a measure of the amount of mRNA from that promoter, the technique is also quantitative. The authors demonstrated the capabilities and efficacy of the technique by using GREM to examine a family of human-specific human endogenous retroviruses (HERVs) called HERV-K in testicular parenchyma samples. Despite some limitations discussed by the authors, the technique successfully identified 54 long terminal repeat elements with promoter activity in the testis, with results showing excellent correlation (R = 0.91) when the proportion of tags found by GREM was compared to expression levels of the same sequences obtained from RT-PCR experiments. –SS

-Buzdin et al. 2006. GREM, a technique for genome-wide isolation and quantitative analysis of promoter active repeats. Nucleic Acids Research 34(9):e67 [Epub ahead of print, May 12, 2006].

How Low Can You Go?

A very low flow rate in liquid chromatography prior to electrospray ionization mass spectrometry can provide significant sensitivity gains. Because decreases in the linear velocity of the mobile phase can cause suboptimal chromatographic performance, a low flow rate should be coupled with a small i.d. column. To date, most LC columns that can be straightforwardly coupled to ESI-MS have inner diameters in the range of 50–75 µm. A joint effort between teams at the Pacific Northwest National Laboratory and Purdue University has now shown that an LC-ESI-MS setup that includes a 10 µm i.d. LC column enables more sensitive and quantitative proteomic analysis than conventional capillary LC.

After being introduced into a microsolid phase extraction (microSPE) precolumn (i.d. of 50 µm), the sample enters the silica-based monolithic 10-µm i.d. LC column, progressing at a flow rate of about 10 nL/min. The setup also includes an integrated ESI emitter, which is pulled to a tip i.d. of about 1 µm. In an initial test, the apparatus was evaluated head-to-head with a conventional LC column having a 150-µm i.d. and a 1.5 µL/min flow rate. As expected, the larger column exhibited ionization suppression effects that compromised peak area measurements for a relatively broad range of typical sample loading amounts. By contrast, the linear response range for the low-flow, 10 µm i.d. column was 9- to 22-fold greater. This suggests that the smaller LC column would be much more appropriate for label-free quantitative proteomic measurements using mass spectrometry, as these methods compare peak intensities to derive relative abundances. In a pilot experiment, Luo et al., show that their method can pick up a 5- to 6-fold difference in the abundance of a protein, as tested by analysis of a FLAG®-tagged protein expressed in a tetra-cycline-regulated manner (and as confirmed by Western blot). The microSPE-LC-ESI-MS arrangement also proved its suitability for a more complex sample, revealing 1332 reliable protein IDs for a 100 ng Shewanella proteome sample. The results should convince many that the overall strategy represents a promising alternative to stable isotope-based quantitative proteomic measurements. –ND

-Luo et al. 2006. More sensitive and quantitative proteomic measurements using very low flow rate porous silica monolithic LC columns with electrospray ionization-mass spectrometry. Journal of Proteome Research 5:1091–1097.

Outside Normal Channels

Microanalysis devices typically employ microfluidics to deliver analytes and mix reactants. Tiny valves and channels reduce solvent and reagent use—compared to traditional reaction vessels—but the need for fluid flow inevitably leads to waste of materials. An alternative to solvent-based payload delivery is proposed by Thomas et al., in a recent paper that builds upon their lab's previous description of magnetic silicon microparticle “chaperones.” The microparticles are prepared from silicon wafers using a multistep process that generates fragments of a porous silicon film. Under appropriate conditions, magnetite nanoparticles can be loaded into the silica microparticles to make them magnetic. However, the magnetite does not saturate the pores in the microparticles, allowing secondary loading with molecules of interest. As a first demonstration that the microparticles could house enzyme payloads, Thomas et al. soaked the particles in an aqueous solution of pronase E. After spectral reflectivity measurements to confirm loading, a single pronase-containing microparticle was immersed in a tiny drop of PBS resting on a solid matrix containing the substrate and an appropriate indicator. Absorbance measurements were used to track enzymatic activity and revealed that a single microparticle could be loaded with nanogram quantities of protein. To test whether the strategy might have potential for replacing microfluidics in reagent delivery, Thomas et al. loaded the microparticles with horseradish peroxidase, an enzyme often used in ELISAs. The microparticles were placed in a dish that also held a 2–4 µL droplet containing HRP substrates. Then, using a small handheld permanent magnet, a single microparticle was transferred to the droplet. Beginning within 2 minutes of delivery, the colorimetric product begins visibly accumulating, revealing that the loaded HRP can diffuse out of the microparticle when it is placed an aqueous solution, and that the enzyme retains its activity. Although higher throughput applications are some ways off, this dry reagent delivery system may find application for a variety of payloads and could liberate microchip researchers from the need for reagent delivery by solvent flow. –ND

(Click to enlarge)

Reprinted with permission. ©2006 The Royal Society of Chemistry.

-Thomas et al. 2006. Delivery of nanogram payloads using magnetic porous silicon microcarriers. Lab on a Chip 6:782–787.

References

1.) Kanatsu-Shinohara,. 2006. Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells. Proceedings of the National Academy of Sciences 103:8018-8023.

2.) Buzdin,. 2006. GREM, a technique for genome-wide isolation and quantitative analysis of promoter active repeats. Nucleic Acids Research [Epub ahead of print, May 12, 2006] 34:e67.

3.) Luo,. 2006. More sensitive and quantitative proteomic measurements using very low flow rate porous silica monolithic LC columns with electrospray ionization-mass spectrometry”. Journal of Proteome Research 5:1091-1097.

4.) Thomas,. 2006. Delivery of nanogram payloads using magnetic porous silicon microcarriers”. Lab on a Chip 6:782-787.