More gene targeting in plants

May 25, 2009

A follow-up to an entry I made a few weeks ago, showing that engineered zinc finger nucleases can be used to target gene insertion in maize.  Abstract and citation without commentary.  Enjoy.

Agricultural biotechnology is limited by the inefficiencies of conventional random mutagenesis and transgenesis. Because targeted genome modification in plants has been intractable1, plant trait engineering remains a laborious, time-consuming and unpredictable undertaking. Here we report a broadly applicable, versatile solution to this problem: the use of designed zinc-finger nucleases (ZFNs) that induce a double-stranded break at their target locus2. We describe the use of ZFNs to modify endogenous loci in plants of the crop species Zea mays. We show that simultaneous expression of ZFNs and delivery of a simple heterologous donor molecule leads to precise targeted addition of an herbicide-tolerance gene at the intended locus in a significant number of isolated events. ZFN-modified maize plants faithfully transmit these genetic changes to the next generation. Insertional disruption of one target locus, IPK1, results in both herbicide tolerance and the expected alteration of the inositol phosphate profile in developing seeds. ZFNs can be used in any plant species amenable to DNA delivery; our results therefore establish a new strategy for plant genetic manipulation in basic science and agricultural applications.

Shukla VK, Doyon Y, Miller JC, DeKelver RC, Moehle EA, Worden SE, Mitchell JC, Arnold NL, Gopalan S, Meng X, Choi VM, Rock JM, Wu YY, Katibah GE, Zhifang G, McCaskill D, Simpson MA, Blakeslee B, Greenwalt SA, Butler HJ, Hinkley SJ, Zhang L, Rebar EJ, Gregory PD, Urnov FD. 2009. Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 459:437-441.


Alternative polyadenylation in development

May 25, 2009

One of the things that is an open book is the true scope and physiological relevance of alternative polyadenylation.  A recent report in PNAS stirs this pot a bit (even if it leaves things still very much up in the air).  Briefly, this group has analyzed various large-scale gene expression repositories – ESTs, SAGE, and microarray – and found a tantalizing possible progression of 3′-UTR length during development.  Specifically, it seems as if global (or average) 3′-UTR length increases during the course of embryogenesis.  This change in the length of 3′-UTRs seems to be due to differential poly(A) site choice.  As I said, very tantalizing.

The abstract and brief commentary follows after the fold.

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Guidelines for quantitative PCR

May 25, 2009

One of the more vexing and difficult issues in molecular biology is the use of PCR as a quantitative assay.  It is easy to find a wide range of approaches to this, and even easier to find papers that refer to “semi-quantitative” PCR.  As one might expect, there is a range of quality in the literature when it comes to these assays.  Worse, though, is the paucity of information that is often provided – things like the numbers of replicates, statistical tools used to analyze the data, and the like.  THis makes it hard to follow many studies, and to replicate the work of others.  (Needless to say, reviewing these papers is a bear.)

There has been a mini-debate of sorts in The Plant Cell over the past few years, and a recent issue has two additions to the discussion.  Follow below the fold for more.

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One year later …

May 25, 2009

Today is the one year anniversary of the beginning of this blog.  I wasn’t sure how things would develop, and still am not.  We’ve had a few good discussions, but what I have noticed most of all is how I get a steady stream of hits on the polyadenylation background essays.  It’s enough to imagine that this blog is sort of an alternative to wikipedia for those looking for information about this process.  This is good, but this interest has not come with questions.  Either these essays are clear (not likely), or someone out there is being shy.  So, those readers who are using this blog as a vehicle to learn about the process, don’t hesitate to ask questions.  You obviously want to learn something about the subject, and the best way to learn is to ask.